Insulin Analogues with an Acyl and Alkylene Glycol Moiety

ABSTRACT

An acylated insulin analogue wherein the insulin analogue comprises a lysine residue connected C-terminally to the A21 amino acid residue or a peptide residue of up to 4 amino acid residues comprising a lysine residue which peptide residue is connected C-terminally to the A21 amino acid residue, characterized in that an acyl moiety comprising an alkylene glycol moiety is attached to the lysine residue in the A22 position or attached to a lysine residue present in the peptide residue that is attached to the C terminal end of the A21 amino acid residue and wherein there is only one lysine (K, Lys) in the insulin analogue, can conveniently be administered pulmonary.

FIELD OF THIS INVENTION

The present invention relates to novel acylated insulin analogues and related aspects thereof.

BACKGROUND OF THIS INVENTION

Insulin is a polypeptide hormone secreted by β-cells of the pancreas. Insulin consists of two polypeptide chains designated the A and B chains which are linked together by two inter-chain disulphide bridges. In human, porcine and bovine insulin, the A and B chains contains 21 and 30 amino acid residues, respectively. However, from species to species, there are variations among the amino acid residues present in the different positions in the two chains. The widespread use of genetic engineering has made it possible to prepare analogues of natural occurring insulins by exchanging one of more of the amino acid residues.

Insulin is used for the treatment of diabetes and diseases connected therewith or resulting from it. Insulin is essential in maintaining normal metabolic regulation. Usually, insulin is administered by injections. Unfortunately, many diabetics are unwilling to undertake intensive therapy due to the discomfort associated with the many injections required to maintain close control of glucose levels. Upon oral administration, insulin is rapidly degraded in the gastro intestinal tract and is not absorbed into the blood stream. Therefore, alternate routes for administering insulin, such as oral, rectal, transdermal, and nasal routes have been investigated. Thus far, however, these routes of administration have not resulted in sufficiently effective insulin absorption.

For decades, both long-acting insulin preparations and fast acting insulin preparations have been available and many patients take 2-4 injections per day. In the last decades, it has turned out that it is extremely important for a diabetic patient to maintain close control of the blood glucose level.

International patent application number having publication number WO 2007/096431 which was published on 30 Aug. 2007 (Novo Nordisk A/S) describes insulins having a complex side chain with no alkylene glycol moieties. International patent application having publication number WO 2006/-082205 (Novo Nordisk A/S) describes insulins having a complex side chain connected to an amino acid in the B chain. According to claim 1, U.S. Pat. No. 6,444,641 B1 relates to a fatty acid-acylated insulin analog comprising an insulin analog to which a fatty acyl chain is joined by an amide bond, wherein said fatty insulin analog has an isoelectric point that is higher than the isoelectric point of insulin. According to claim 1, WO 2006/082205 (Novo Nordisk NS) relates to insulin derivatives having a side chain attached either to the α-amino group of the N-terminal amino acid residue of B chain or to an ε-amino group of a Lys residue present in the B chain of the parent insulin molecule. Pharm. Res. 21 (2004), 1498-1504, deals with the mechanism of protraction of insulin detemir.

DEFINITIONS

Herein, the term insulin covers natural occurring insulins, e.g., human insulin, as well as insulin analogues.

Herein, the term amino acid residue covers an amino acid from which a hydrogen atom has been removed from an amino group and/or a hydroxy group has been removed from a carboxy group and/or a hydrogen atom has been removed from a mercapto group. Imprecise, an amino acid residue may be designated an amino acid.

Herein, the term peptide residue covers a peptide from which a hydrogen atom has been removed from an amino group and/or a hydroxy group has been removed from a carboxy group and/or a hydrogen atom has been removed from a mercapto group. Imprecise, a peptide residue may be designated a peptide.

Herein, the term insulin analogue covers a polypeptide which has a molecular structure which formally can be derived from the structure of a naturally occurring insulin, e.g., human insulin, by deleting and/or substituting (replacing) one or more amino acid residue occurring in the natural insulin and by adding one or more amino acid residues to the A21 amino acid residue. Preferably, the added and/or substituted amino acid residues are codable amino acid residues. For example, the A chain may be extended at its C-terminal end, e.g., by 1, 2, 3 or 4 amino acid residues (compared with human insulin) the positions of which are denoted A22, A23, A24 and A25, respectively. Even though the insulin analogue has an extension at the A21/A22 position, there may be deletions at other positions in said insulin analogue. Similarly as in human insulin, in the insulin analogue of this invention, the A21 amino acid residue is connected to a Cys residue in the 20 position which Cys residue participates in the forming of an inter-chain disulphide bridge. Herein, also the term parent insulin or parent insulin analogue is used for the insulin analogue. Mainly, the term parent is used when differentiating from an insulin analogue carrying a side chain which, for example, can be introduced chemically by acylation.

Herein terms like A1, A2, A3 etc. indicates the position 1, 2 and 3, respectively, in the A chain of insulin (counted from the N-terminal end). Similarly, terms like B1, B2, B3 etc. indicates the position 1, 2 and 3, respectively, in the B chain of insulin (counted from the N-terminal end). Using the one letter codes for amino acids, terms like A21A, A21G and A21Q designates that the amino acid in the A21 position is A, G and Q, respectively. Using the three letter codes for amino acids, the corresponding expressions are AlaA21, GlyA21 and GlnA21, respectively.

Herein terms like desB29 and desB30 indicate an insulin analogue lacking the B29 or B30 amino acid residue, respectively.

The numbering of the positions in insulin analogues and A and B chains is done so that the parent compound is human insulin with the numbering used for it.

Herein, the expression “codable” in connection with terms like amino acid, amino acid residue, peptide or peptide residue is used to indicate an amino acid, amino acid residue, peptide or peptide residue which can be coded for by a triplet (“codon”) of nucleotides, vide genetic engineering.

Herein, the term mutation covers any change in amino acid sequence (substitutions and insertions with codable amino acids as well as deletions).

With fast acting insulin or bolus insulin is meant an insulin having a fast onset of action similar to or faster than regular human insulin and/or a duration of action that is similar to or only slightly longer than that of regular human insulin.

With prolonged acting insulin or basal insulin is meant an insulin having a longer duration of action than normal or regular human insulin.

By high physical stability is meant a tendency to fibrillation being less than 50% of that of human insulin. Fibrillation may be described by the lag time before fibril formation is initiated at a given conditions.

A polypeptide with insulin receptor and IGF-1 receptor affinity is a polypeptide which is capable of interacting with an insulin receptor and a human IGF-1 receptor in a suitable binding assay. Such receptor assays are well-know within the field and are further described in the examples. The acylated insulin analogues of this invention will not bind to the IGF-1 receptor or will have a rather low affinity to said receptor. More precisely, the acylated insulin analogues of this invention will have an affinity towards the IGF-1 receptor of substantially the same magnitude or less as that of human insulin.

For the sake of convenience, here follows the names of amino acids with the usual three letter codes & one letter codes in parenthesis: Glycine (Gly & G), proline (Pro & P), alanine (Ala & A), valine (Val & V), leucine (Leu & L), isoleucine (Ile & I), methionine (Met & M), cysteine (Cys & C), phenylalanine (Phe & F), tyrosine (Tyr & Y), tryptophan (Trp & W), histidine (His & H), lysine (Lys & K), arginine (Arg & R), glutamine (Gln & Q), asparagine (Asn & N), glutamic acid (Glu & E), aspartic acid (Asp & D), serine (Ser & S) and threonine (Thr & T). If, due to typing errors, there are deviations from the commonly used codes, the commonly used codes apply. The amino acids present in the insulins of this invention are, preferably, amino acids which can be coded for by a nucleic acid. Amino acids like Glu and Asp can be in the α, γ, L or D form.

The following abbreviations have been used in the specification and examples: Da is Dalton (molecular weight), kDa is kilo-Dalton (=1000 Da), Mw is molecular weight, OSu is 1-succinimidyloxy=2,5-dioxopyrrolidin-1-yloxy, RT is room temperature, SA is sinapinic acid and Su is 1-succinimidyl=2,5-dioxopyrrolidin-1-yl, DCM is dichloromethane, DIEA (and DIPEA) is N,N-diisopropylethylamine, TSTU is N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate, Tris is tris(hydroxymethyl)aminomethane, CV is column volume, NMP is N-methylpyrrolidin-2-one, OTBU is tert-butoxy, OEG is the amino acid 8-amino-3,6-dioxaoctanoic acid, HSA is human serum albumin, TNBS is 2,4,6-trinitrobenzenesulfonic acid, HOBT (or HOBt) is 1-hydroxybenzotriazole, HOAt is 1-hydroxy-7-aza-benzotriazole, NaOH is sodium hydroxide, DMF is N,N-dimethyl formamide, THF is tetrahydrofuran, TFA is trifluoroacetic acid, mmol is millimoles, Fmoc is fluoren-9-ylmethoxycarbonyl, OEG is 8-amino-3,6-dioxaoctanoic acid (or a residue thereof), gGlu (herein also designated γGlu) is gamma-glutamic acid, and in example 48, for convenience, the following annotations are used to specify the sequence of the acyl moieties of the insulins of the invention and of the prior art: C18 is octadecanedioyl, C20 is eicosanedioyl, gGlu is gamma-glutamic acid, PEG3 is 3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionic acid, PEG5 is 3-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)ethoxy]ethoxy}propionic acid and PEG7 is 3-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)ethoxy]ethoxy}ethoxy)ethoxy]propionic acid (the complete structures/sequences can be found in the examples).

OBJECTS OF THIS INVENTION

The object of this invention is to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

One object of this invention is to furnish insulin derivatives, which have good bioavailability.

Another object of the invention is to furnish insulin derivatives, which are usable as fast acting insulins.

Another object of this invention is to furnish insulin derivatives, which can be administered pulmonary.

Another object of this invention is to furnish insulin derivatives, which have a prolonged action profile.

Another object of this invention is to furnish insulin derivatives, which are usable as basal insulins.

Another object of this invention is to furnish insulin derivatives, which have high insulin receptor binding affinities.

Another aspect of this invention is to improve the in vivo half-life of insulins.

Another object of this invention is to furnish insulin derivatives having a lover tendency to give rise to hypoglycaemic conditions than human insulin has.

Another aspect of this invention is to find insulins having a satisfactory physical stability, especially after storage for 2 years.

Another aspect of this invention is to find insulins having a satisfactory chemical stability, especially after storage for 2 years.

Another aspect of this invention is to find insulins having a satisfactory proteolytic stability, especially after storage for 2 years.

Another aspect of this invention is to find insulins having a satisfactory solubility.

SUMMARY OF THIS INVENTION

Briefly, this invention relates to an acylated insulin analogue wherein the insulin analogue comprises an amino acid residue connected to the C terminal end of the A21 amino acid residue or a peptide residue of up to 4 amino acid residues connected to the C terminal end of the A21 amino acid residue, characterized in that an acyl moiety comprising an alkylene glycol moiety is attached to a lysine residue in the A22 position or attached to a lysine residue present in the peptide residue attached to the C terminal end of the A21 amino acid residue. Hence, the insulin analogue comprises an amino acid in the A22 position.

The insulins of the invention (acylated insulin analogues comprising one or more alkylene glycol moieties) have surprisingly been found to posses higher insulin receptor binding affinities than similar acylated insulin analogues without the alkylene glycol moieties, when measured in insulin receptor binding assays conducted in presence of high HSA (human serum albumin) concentrations, eg. 4.5% HSA.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Surprisingly, the acylated insulin analogues of this invention show a good bioavailability and high insulin receptor binding affinity in presence of high HSA concentrations.

The insulin analogue present in an acylated insulin analogue according to this invention can formally be illustrated as human insulin which has one amino acid residue connected C-terminally to the amino acid residue in position A21 or as human insulin which has a peptide residue of up to 4 amino acid residues connected C-terminally to the amino acid residue in position A21 and, optionally, wherein one or more of the amino acid residues in positions A1-A21 and B1-B30 has been deleted or substituted by another amino acid residue. As to nomenclature, the amino acid residue connected to the amino acid residue in position A21 is in the A22 position. Similarly, the amino acid residues present in the peptide residue connected to the amino acid residue in position A21 is in the positions A22, A23, A24 or A25.

The acylated insulin analogues of this invention can, formally be build from an insulin analogue and an alkylene glycol containing acyl moiety having the collective formula (I): Acy-AA1_(n)-AA2_(m)-AA3_(p), wherein Acy, AA1, AA2, AA3, n, m and p are as defined herein, e.g., by formally removing a hydrogen atom from an amino group in the insulin analogue and attaching the side chain of formula (I) thereto.

In the acylated insulin analogues of this invention, the alkylene glycol containing acyl moiety of formula (I) is connected to a lysine residue in a position which is on the C terminal side or end of the A21 amino acid residue. For example, the alkylene glycol containing acyl moiety of formula (I) may be connected to a lysine residue in position A22. If the alkylene glycol containing acyl moiety of formula (I) is connected to a peptide residue which is connected to the amino acid residue in position A21, the alkylene glycol containing acyl moiety of formula (I) is connected to a lysine residue in any of the positions A22, A23, A24, or A25.

As mentioned herein, the acyl group present in the formula Acy-AA1_(n)-AA2_(m)-AA3_(p)- originates from a fatty acid or a fatty diacid.

Herein, the term “fatty acid” covers a linear or branched, aliphatic carboxylic acids having at least two carbon atoms and being saturated or unsaturated. Non limiting examples of fatty acids are myristic acid, palmitic acid, and stearic acid.

Herein, the term “fatty diacid” covers a linear or branched, aliphatic dicarboxylic acids having at least two carbon atoms and being saturated or unsaturated. Non limiting examples of fatty diacids are succinic acid, hexanedioic acid, octanedioic acid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid, heptadecanedioic acid, octadecanedioic acid, and eicosanedioic add.

The neutral cyclic amino acid residue designated AA1 is an amino acid containing a saturated 6-membered carbocyclic ring, optionally containing a nitrogen hetero atom, and preferably the ring is a cyclohexane ring or a piperidine ring. Preferably, the molecular weight of this neutral cyclic amino acid is in the range from about 100 to about 200 Da.

The acidic amino acid residue designated AA2 is an amino acid with a molecular weight of up to about 200 Da comprising two carboxylic acid groups and one primary or secondary amino group.

The neutral, alkyleneglycol-containing amino acid residue designated AA3 is an alkyleneglycol moiety, optionally an oligo- or polyalkyleneglycol moiety containing a carboxylic acid functionality at one end and a amino group functionality at the other end.

Herein, the term alkyleneglycol moiety covers oligo- and polyalkyleneglycol moieties as well as monoalkyleneglycol moieties. Polyalkyleneglycols comprises polyethyleneglycol based, polypropyleneglycol based and polybutyleneglycol based chains, i.e., chains that are based on the repeating unit —CH₂CH₂O—, —CH₂CH₂CH₂O— or —CH₂CH₂CH₂CH₂O—. The alkyleneglycol moiety can be monodisperse (with well defined length/molecular weight) as well as polydisperse (with less well defined length/average molecular weight). Monoalkyleneglycol moieties comprises —OCH₂CH₂O—, —OCH₂CH₂CH₂O— or —OCH₂CH₂CH₂CH₂O— containing different groups at each end.

As mentioned herein, the order by which AA1, AA2 and AA3 appears in the acyl moiety with the formula (I) (Acy-AA1_(n)-AA2_(m)-AA3_(p)-) can be interchanged independently. Consequently, the formula Acy-AA1_(n)-AA2_(m)-AA3_(p)- also covers moieties like, e.g., the formula Acy-AA2_(m)-AA1_(n)-AA3_(p)- and the formula Acy-AA3_(p)-AA2_(m)-AA1_(n)-, wherein Acy, AA1, AA2, AA3, n, m and p are as defined herein.

As mentioned herein, the connections between the moieties Acy, AA1, AA2 and/or AA3 are amide bonds (—CONH— wherein —CO— originates from one of the moieties Acy, AA1, AA2 and AA3 and —NH— originates from one of the moities AA1, AA2 and AA3).

PREFERRED EMBODIMENTS OF THE INVENTION

In an embodiment, the parent insulin of the invention can, apart from the lysine in position A22 and apart from any peptide residue of up to 4 amino acids connected C terminally to the A21 amino acid residue and apart from the B29R, and/or desB30 mutations, comprise one or more of the following mutations. Here, first the position in the A or B chain is given and, thereafter, the possible amino acid residue(s) is given as the one letter codes. In one embodiment, there are 5 mutations, in another embodiment, there are 4 mutations, in another embodiment, there are 3 mutations, in another embodiment, there are 2 mutations, in another embodiment, there is 1 mutation and in another embodiment, there is no mutation, apart from the lysine in position A22 and apart from any peptide residue of up to 4 amino acids connected C terminally to the A21 amino acid residue and apart from the B29R, and/or desB30 mutations:

A4: A or Q.

A5: L.

A8: R, N, Q, E, H, L or W.

A9: R or L.

A14: E or D.

A15: A or T.

A16: M.

A17: D or F.

A18: R, L, I, D, or V.

A21: G or A.

B3: A, R, H, I, L, M, F, W, Y, S or T.

B10: D or E.

B25: Y, H or desB25.

B26: Q, E, S or desB26.

B27: H, L, M, W or Y.

B28: D or E.

In another preferred embodiment the parent insulin of the invention comprise the A4A or A4Q mutations.

In another preferred embodiment the parent insulin of the invention comprises the A5L mutation.

In another preferred embodiment the parent insulin of the invention comprises the A8L, ABN, A8Q, A8E, A8H, A8L, or A8W mutation. In another preferred embodiment the parent insulin of the invention comprises the A8H mutation.

In another preferred embodiment the parent insulin of the invention comprises the A9R or A9L mutation. In another preferred embodiment the parent insulin of the invention comprises the A9L mutation.

In another preferred embodiment the parent insulin of the invention comprises the A14E or A14D mutation.

In another preferred embodiment the parent insulin of the invention comprises the A15A or A15T mutation.

In another preferred embodiment the parent insulin of the invention comprises the A16M mutation.

In another preferred embodiment the parent insulin of the invention comprises the A17D or A17F mutation.

In another preferred embodiment the parent insulin of the invention comprises the A18R, A18L, A18I, A18D, or A18V mutation. In another preferred embodiment the parent insulin of the invention comprises the A18L or A18V mutation. In another preferred embodiment the parent insulin of the invention comprises the A18I mutation.

In another preferred embodiment the parent insulin of the invention comprises the A21G or A21A mutation.

In another preferred embodiment the parent insulin of the invention comprises the B3A, B3R, B3H, B3I, B3L, B3M, B3F, B3W, B3Y, B3S or B3T mutation.

In another preferred embodiment the parent insulin of the invention comprises the B10D or B10E mutation.

In another preferred embodiment the parent insulin of the invention comprises the B25Y, B25H, or desB25 mutation.

In another preferred embodiment the parent insulin of the invention comprises the B26Q, B26E, B26S, or desB26 mutation.

In another preferred embodiment the parent insulin of the invention comprises the B27H, B27L, B27M, B27W, or B27Y mutation. In another preferred embodiment the parent insulin of the invention comprises the B27W or B27Y mutation.

In another preferred embodiment the parent insulin of the invention comprises the B28D or B28E mutation.

In another preferred embodiment the parent insulin of the invention comprises the A21 Q, B1Q, desB1, B3Q, B3S, B3T, B13Q, or desB27 mutation.

Non-limiting specific examples of parent insulin analogues which may be present in the acylated insulin analogues of this invention comprise the following where deviations from human insulin are given:

A22K, B29R, desB30 A14E, A22K, B25H, B29R, desB30 A8H, A22K, B29R, desB30 A18L, A22K, B29R, desB30 A18I, A22K, B29R, desB30 A18D, A22K, B29R, desB30 A5L, A22K, B29R, desB30 A8H, A18L, A22K, B29R, desB30 A8H, A18L, A22K, B10E, B29R, desB30 A5L, A8H, A17D, A18L, A22K, B10E, B29R, desB30 A8H, A22K, B10E, B29R, desB30 A5L, A8H, A17D, A22K, B10E, B29R, desB30 A22K, B27Y, B29R, desB30 A9L, A22K, B29R, desB30 A4A, A22K, B29R, desB30 A4Q, A22K, B29R, desB30 A16L, A22K, B29R, desB30 A17F, A22K, B29R, desB30 A17D, A22K, B29R, desB30 A18V, A22K, B29R, desB30 A22K, B10D, B29R, desB30 A22K, B10E, B29R, desB30 A22K, B27W, B29R, desB30 A22K, B27Y, B29R, desB30 A22K, B28E, B29R, desB30 A22K, B27Y, B28E, B29R, desB30 A8H, A22K, B28E, B29R, desB30 A22K, desB26, B28E, B29R, desB30 A22K, desB25, B29R, desB30 A22K, desB26, B29R, desB30 A22K, B28E, B29R, desB30 A22K, B28D, B29R, desB30 A9L, A22K, B28E, B29R, desB30 A4A, A22K, B28E, B29R, desB30 A4Q, A22K, B28E, B29R, desB30 A5L, A22K, B28E, B29R, desB30 A16L, A22K, B28E, B29R, desB30 A17F, A22K, B28E, B29R, desB30 A17D, A22K, B28E, B29R, desB30 A18V, A22K, B28E, B29R, desB30 A22K, B10D, B28E, B29R, desB30 A22K, B10E, B28E, B29R, desB30 A22K, B27W, B28E, B29R, desB30 A22G, A23K, B29R, desB30 A22G, A23G, A24K, B29R, desB30 A22G, A23G, A24G, A25K, B29R, desB30 A21Q, A22G, A23G, A24K, B29R, desB30 A21G, A22G, A23G, A24K, B29R, desB30 A21Q, A22G, A23G, A24G, A25K, B29R, desB30 A21G, A22G, A23G, A24G, A25K, B29R, desB30 A21G, A22K, B29R, desB30 human insulin A21G, A22G, A23K, B29R, desB30 A21G, A22G, A23G, A24K, B29R, desB30 A21G, A22G, A23G, A24G, A25K, B29R, desB30 A21Q, A22K, B29R, desB30 A21Q, A22G, A23K, B29R, desB30 A21Q, A22G, A23G, A24K, B29R, desB30 A21Q, A22G, A23G, A24G, A25K, B29R, desB30 A14E, A21Q, A22K, B25H, B29R, desB30 A14E, A21G, A22K, B25H, B29R, desB30 A14E, A21Q, A22G, A23K, B25H, B29R, desB30 A14E, A21G, A22G, A23K, B25H, B29R, desB30 A14E, A21Q, A22G, A23G, A24K, B25H, B29R, desB30 A14E, A21G, A22G, A23G, A24K, B25H, B29R, desB30 A14E, A21Q, A22G, A23G, A24G, A25K, B25H, B29R, desB30 A14E, A21G, A22G, A23G, A24G, A25K, B25H, B29R, desB30 A22K, B3Q, B29R, desB30 A22K, B3S, B29R, desB30 A22K, B3T, B29R, desB30 A22K, B1Q, B29R, desB30 A18Q, A22K, B29R, desB30 A22K, desB1, B3Q, B29R, desB30 A22K, B28D, B29R, desB30 A22K, desB27, B28E, B29R, desB30 A22K, B28R, desB29, desB30 A22K, B3Q, B28E, B29R, desB30 A22K, B13Q, B29R, desB30 A22K, desB1, B29R, desB30 A21Q, A22G, A23K, B29R, desB30 A21Q, A22G, A23G, A24K, B29R, desB30 A21Q, A22G, A23G, A24G, A25K, B29R, desB30 A21A, A22K, B29R, desB30 A21A, A22G, A23K, B29R, desB30 A21G, A22G, A23K, B29R, desB30 A21A, A22G, A23G, A24K, B29R, desB30 A21G, A22G, A23G, A24K, B29R, desB30 A21G, A22G, A23G, A24G, A25K, B29R, desB30 A21A, A22G, A23G, A24G, A25K, B29R, desB30 A21Q, A22K, B3Q, B29R, desB30 A21A, A22K, B3Q, B29R, desB30 A21G, A22K, B3Q, B29R, desB30

Non-limiting specific examples of the acyl moieties of the formula Acy-AA1_(n)-AA2_(m)-AA3_(p)- which may be present in the acylated insulin analogues of this invention are the following:

In one embodiment, the moiety designated AA3 is elected among the following compounds from which a hydrogen atom has been removed from the amino group and a hydroxy group has been removed from the carboxy group:

Any of the above non-limiting specific examples of acyl moieties of the formula Acy-AA1_(n)-AA2_(m)-AA3_(p)- can be attached to an epsilon amino group of a lysine residue present in any of the above non-limiting specific examples of insulin analogues thereby giving further specific examples of acylated insulin analogues of this invention.

The parent insulins can be prepared in a manner known per se. For example, they can be produced by expressing a DNA sequence encoding the single-chain insulin in question in a suitable host cell by well known technique as disclosed in e.g., EP 1,246,845. The insulin is expressed in a transformed host cell as a precursor molecule which is converted into the desired insulin molecule by enzymatic and chemical in vitro processes as disclosed in, e.g., EP 163,529 and EP 214,826. The precursor molecule may be expressed with an N-terminal extension which is later cleaved of as disclosed in, e.g., EP 1246,845. Examples of N-terminal extensions of the type suitable in the present invention are, e.g., disclosed in U.S. Pat. No. 5,395,922 and EP patent No. 765,395. More specifically, reference can be made to WO 2006/082205, from page 37, line 31, to page 39, line 29.

The parent insulin analogue can be converted into the acylated insulin analogue of this invention by introducing of the desired group of the formula Acy-AA1_(n)-AA2_(m)-AA3_(p)- in the lysine residue in the insulin analogue. The desired group of the formula Acy-AA1_(n)-AA2_(m)-AA3_(p)- can be introduced by any convenient method and many methods are disclosed in the prior art for such reactions. More details appear from the following examples.

Acylated insulin analogues of this invention may be provided in the form of essentially zinc free compounds or in the form of zinc complexes. When zinc complexes of an acylated insulin analogue of this invention are provided, two Zn²⁺ ions, three Zn²⁺ ions or four Zn²⁺ ions can be bound to each insulin hexamer. Solutions of zinc complexes of the acylated insulin analogues of this will contain mixtures of such species.

In a further aspect, this invention relates to a pharmaceutical composition comprising a therapeutically effective amount of an acylated insulin analogue of this invention together with a pharmaceutically acceptable carrier which can be used for the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in patients in need of such a treatment. An acylated insulin analogue of this invention can be used for the manufacture of a pharmaceutical composition for use in the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia. Such compositions are prepared in a manner known per se.

In a further aspect of this invention, there is provided a pharmaceutical composition for treating type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in a patient in need of such a treatment, comprising a therapeutically effective amount of an acylated insulin analogues of this invention in mixture with an insulin or an insulin analogue which has a rapid onset of action, together with pharmaceutically acceptable carriers and additives.

In a further aspect, this invention relates to a pulmonary application for treating type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in a patient in need of such a treatment, comprising a therapeutically effective amount of an acylated insulin analogues of this invention, optionally in mixture with an insulin or an insulin analogue which has a rapid onset of action, together with pharmaceutically acceptable carriers and additives.

In one aspect, this invention provides a pharmaceutical composition being a mixture of an acylated insulin analogue of this invention and a rapid acting insulin analogue selected from the group consisting of AspB28 human insulin; LysB28 ProB29 human insulin and LysB3 GluB29 human insulin.

The acylated insulin analogues of this invention and the rapid acting insulin analogue can be mixed in a ratio of about 90/10%; about 70/30% or about 50/50%.

In a further aspect of the invention, there is provided a method of treating type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in a patient in need of such a treatment, comprising administering to the patient a therapeutically effective amount of an acylated insulin analogues of this invention together with a pharmaceutically acceptable carrier and pharmaceutical acceptable additives.

In a further aspect of the invention, there is provided a method of treating type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in a patient in need of such a treatment, comprising administering to the patient a therapeutically effective amount of an acylated insulin analogues of this invention in mixture with an insulin or an insulin analogue which has a rapid onset of action, together with a pharmaceutically acceptable carrier and pharmaceutical acceptable additives.

In a further aspect of the invention there is provided a pharmaceutical composition for the treatment of diabetes in a patient in need of such treatment, comprising a therapeutically effective amount of an acylated insulin analogues of this invention in mixture with an insulin or an insulin analogue which has a rapid onset of action, together with a pharmaceutically acceptable carrier.

In a further aspect of the invention there is provided a pharmaceutical composition according to the invention intended for pulmonal administration.

In a further aspect of the invention there is provided a method of treating diabetes in a patient in need of such a treatment, comprising administering to the patient a therapeutically effective amount of an acylated insulin analogues according to claim 1 together with a pharmaceutically acceptable carrier.

In a further aspect of the invention there is provided a method of treating diabetes in a patient in need of such a treatment, comprising administering to the patient a therapeutically effective amount of an acylated insulin analogues according to claim 1 in mixture with an insulin or an insulin analogue which has a rapid onset of action, together with a pharmaceutically acceptable carrier.

In a further aspect, the present invention relates to acylated insulins of this invention which have insulin receptor binding affinities as described herein, measured in presence of HSA (for example in presence of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 4.5% HSA), that are >3%, more preferred >5%, more preferred >10%, more preferred >15%, more preferred >20%, more preferred >30%, more preferred >40%, more preferred >50%, more preferred >60% measured relative to human insulin.

In a further aspect, the present invention relates to acylated insulin analogues of this invention which have an overall hydrophobicity which is essentially similar to that of human insulin.

In a further aspect, the acylated insulin analogues of this invention have a hydrophobic index, k′_(rel), which is in the range from about 0.02 to about 10, from about 0.1 to about 5; from about 0.5 to about 5; from about 0.2 to about 2; from about 0.2 to about 1; from about 0.1 to about 2; or from about 0.5 to about 2. The hydrophobicity (hydrophobic index) of the acylated insulin analogues of this invention relative to human insulin, k′_(rel), was measured on a LiChrosorb RP18 (5 μm, 250×4 mm) HPLC column by isocratic elution at 40° C. using mixtures of A) 0.1 M sodium phosphate buffer, pH 7.3, containing 10% acetonitrile, and B) 50% acetonitrile in water as eluents. The elution was monitored by following the UV absorption of the eluate at 214 nm. Void time, t_(o), was found by injecting 0.1 mM sodium nitrate. Retention time for human insulin, t_(human), was adjusted to at least 2t₀ by varying the ratio between the A and B solutions. k′_(rel)=(t_(derivative)−t₀)/(t_(human)−t₀).

In another aspect, the invention relates to a pharmaceutical composition comprising an acylated insulin analogue of this invention which is soluble at physiological pH values.

In another aspect, the invention relates to a pharmaceutical composition comprising an acylated insulin analogue of this invention which is soluble at pH values in the interval from about 6.5 to about 8.5.

In another aspect, the invention relates to a pharmaceutical composition with a prolonged profile of action which comprises an acylated insulin analogue of this invention.

In another aspect, the invention relates to insulins with hepatoselective or hepatopreferential action.

In another aspect, the invention relates to a pharmaceutical composition which is a solution containing from about 120 nmol/ml to about 2400 nmol/ml, from about 400 nmol/ml to about 2400 nmol/ml, from about 400 nmol/ml to about 1200 nmol/ml, from about 600 nmol/ml to about 2400 nmol/ml, or from about 600 nmol/ml to about 1200 nmol/ml of an acylated insulin analogues of this invention or of a mixture of an acylated insulin analogues of this invention together with a rapid acting insulin analogue.

Use of the Compounds of this Invention

The route of administration may be any route which effectively transports a compound of this invention to the desired or appropriate place in the body, such as parenterally, for example, subcutaneously, intramuscularly or intraveneously. Alternatively, a compound of this invention can be administered orally, pulmonary, or nasally.

For parenterally administration, a compound of this invention is formulated analogously with the formulation of known insulins. Furthermore, for parenterally administration, a compound of this invention is administered analogously with the administration of known insulins and the physicians are familiar with this procedure.

Parenteral administration can be performed by means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump.

Injectable compositions of a compound of this invention can be prepared using the conventional techniques of the pharmaceutical industry which involve dissolving and mixing the ingredients as appropriate to give the desired end product. Thus, according to one procedure, a compounds of this invention is dissolved in an amount of water which is somewhat less than the final volume of the composition to be prepared. An isotonic agent, a preservative and a buffer is added as required and the pH value of the solution is adjusted—if necessary—using an acid, for example, hydrochloric acid, or a base, for example, aqueous sodium hydroxide, as needed. Finally, the volume of the solution is adjusted with water to give the desired concentration of the ingredients.

More precisely, an insulin preparation of this invention, for example a solution or suspension, may be prepared by dissolving a compound of this invention in an aqueous medium at slightly acidic conditions, for example, in a concentration in the range from about 240 to about 1200 nmole/ml. The aqueous medium is made isotonic, for example, with sodium chloride or glycerol. Furthermore, the aqueous medium may contain zinc ions in a concentrations of up to about 20 μg of Zn⁺⁺ per unit of insulin activity, buffers such as acetate and citrate and preservatives such as m-cresol or phenol. The pH value of the solution is adjusted towards neutrality without getting too close to the isoelectric point of the compound of this invention in order to avoid precipitation. The pH value of the final insulin preparation depends upon the number of charges that, optionally, have been changed in the compound of this invention, the concentration of zinc ions, the concentration of the compound of this invention and the compound of this invention selected. The insulin preparation is made sterile, for example, by sterile filtration.

The insulin preparations of this invention are used similarly to the use of the known insulin preparations.

The amount of a compound of this invention to be administered, the determination of how frequently to administer a compound of this invention, and the election of which compound or compounds of this invention to administer, optionally together with another antidiabetic compound, is decided in consultation with a practitioner who is familiar with the treatment of diabetes.

Hence, this invention also relates to a method of treating diabetes, comprising administering an affective amount of a compound of this invention to a patient in need of such treatment.

Pharmaceutical Compositions

The acylated insulin analogues of this invention may be administered subcutaneously, orally, or pulmonary.

For subcutaneous administration, the acylated insulin analogues of this invention are formulated analogously with the formulation of known insulins. Furthermore, for subcutaneous administration, the acylated insulin analogues of this invention are administered analogously with the administration of known insulins and, generally, the physicians are familiar with this procedure.

Acylated insulin analogues of this invention may be administered by inhalation in a dose effective to increase circulating insulin levels and/or to lower circulating glucose levels. Such administration can be effective for treating disorders such as diabetes or hyperglycemia. Achieving effective doses of insulin requires administration of an inhaled dose in the range from about 0.5 μg/kg to about 50 μg/kg of an acylated insulin analogue of this invention. A therapeutically effective amount can be determined by a knowledgeable practitioner, who will take into account factors including insulin level, blood glucose levels, the physical condition of the patient, the patient's pulmonary status, or the like.

The acylated insulin analogues of this invention may be delivered by inhalation to achieve slow absorption and/or reduced systemical clearance thereof. Different inhalation devices typically provide similar pharmacokinetics when similar particle sizes and similar levels of lung deposition are compared.

The acylated insulin analogues of this invention may be delivered by any of a variety of inhalation devices known in the art for administration of a therapeutic agent by inhalation. These devices include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Preferably, the acylated insulin analogues of this invention are delivered by a dry powder inhaler or a sprayer. There are several desirable features of an inhalation device for administering acylated insulin analogues of this invention. For example, delivery by the inhalation device is advantageously reliable, reproducible, and accurate. The inhalation device should deliver small particles or aerosols, e.g., less than about 10 μm, for example about 1-5 μm, for good respirability. Some specific examples of commercially available inhalation devices suitable for the practice of this invention are Cyclohaler, Turbohaler™ (Astra), Rotahaler® (Glaxo), Diskus® (Glaxo), Spiros™ inhaler (Dura), devices marketed by Inhale Therapeutics, AERx™ (Aradigm), the Ultravent® nebulizer (Mallinckrodt), the Acorn II® nebulizer (Marquest Medical Products), the Ventolin® metered dose inhaler (Glaxo), the Spinhaler® powder inhaler (Fisons), or the like.

As those skilled in the art will recognize, the formulation of acylated insulin analogues of this invention, the quantity of the formulation delivered and the duration of administration of a single dose depend on the type of inhalation device employed. For some aerosol delivery systems, such as nebulizers, the frequency of administration and length of time for which the system is activated will depend mainly on the concentration of acylated insulin analogues in the aerosol. For example, shorter periods of administration can be used at higher concentrations of acylated insulin analogues in the nebulizer solution. Devices such as metered dose inhalers can produce higher aerosol concentrations, and can be operated for shorter periods of time to deliver the desired amount of an acylated insulin analogue of this invention. Devices such as powder inhalers deliver active agent until a given charge of agent is expelled from the device. In this type of inhaler, the amount of insulin acylated insulin analogues of this invention in a given quantity of the powder determines the dose delivered in a single administration.

The particle size of acylated insulin analogues of this invention in the formulation delivered by the inhalation device is critical with respect to the ability of insulin to make it into the lungs, and preferably into the lower airways or alveoli. Preferably, the acylated insulin analogues of this invention is formulated so that at least about 10% of the acylated insulin analogues delivered is deposited in the lung, preferably about 10 to about 20%, or more. Particles of the acylated insulin analogue delivered by inhalation have a particle size preferably less than about 10 μm, more preferably in the range of about 1 μm to about 5 μm. The formulation of the acylated insulin analogue is selected to yield the desired particle size in the chosen inhalation device.

Advantageously for administration as a dry powder, an acylated insulin analogue of this invention is prepared in a particulate form with a particle size of less than about 10 μm, preferably about 1 to about 5 μm. The preferred particle size is effective for delivery to the alveoli of the patient's lung. Preferably, the dry powder is largely composed of particles produced so that a majority of the particles have a size in the desired range. Advantageously, at least about 50% of the dry powder is made of particles having a diameter less than about 10 μm. Such formulations can be achieved by spray drying, milling, micronisation, or critical point condensation of a solution containing an acylated insulin analogue of this invention and other desired ingredients. Other methods also suitable for generating particles useful in the current invention are known in the art.

The particles are usually separated from a dry powder formulation in a container and then transported into the lung of a patient via a carrier air stream. Typically, in current dry powder inhalers, the force for breaking up the solid is provided solely by the patient's inhalation. In another type of inhaler, air flow generated by the patient's inhalation activates an impeller motor which deagglomerates the particles.

Formulations of acylated insulin analogues of this invention for administration from a dry powder inhaler typically include a finely divided dry powder containing the derivative, but the powder can also include a bulking agent, carrier, excipient, another additive, or the like. Additives can be included in a dry powder formulation of an acylated insulin analogue, e.g., to dilute the powder as required for delivery from the particular powder inhaler, to facilitate processing of the formulation, to provide advantageous powder properties to the formulation, to facilitate dispersion of the powder from the inhalation device, to stabilize the formulation (for example, antioxidants or buffers), to provide taste to the formulation, or the like. Advantageously, the additive does not adversely affect the patient's airways. The acylated insulin analogue can be mixed with an additive at a molecular level or the solid formulation can include particles of an acylated insulin analogue mixed with or coated on particles of the additive. Typical additives include mono-, di-, and polysaccharides; sugar alcohols and other polyols, such as, e.g., lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol, starch, or combinations thereof; surfactants, such as sorbitols, diphosphatidyl choline, or lecithin; or the like. Typically an additive, such as a bulking agent, is present in an amount effective for a purpose described above, often at about 50% to about 90% by weight of the formulation. Additional agents known in the art for formulation of a protein such as insulin analogue protein can also be included in the formulation.

A spray including the acylated insulin analogues of this invention can be produced by forcing a suspension or solution of an acylated insulin analogue through a nozzle under pressure. The nozzle size and configuration, the applied pressure, and the liquid feed rate can be chosen to achieve the desired output and particle size. An electrospray can be produced, e.g., by an electric field in connection with a capillary or nozzle feed. Advantageously, particles of insulin conjugate delivered by a sprayer have a particle size less than about 10 μm, preferably in the range of about 1 μm to about 5 μm.

Formulations of acylated insulin analogues of this invention suitable for use with a sprayer will typically include the acylated insulin analogues in an aqueous solution at a concentration of from about 1 mg to about 500 mg of an acylated insulin analogue per ml of solution. Depending on the acylated insulin analogue chosen and other factors known to the medical advisor, the upper limit may be lower, e.g., 450, 400, 350, 300, 250, 200, 150, 120, 100 or 50 mg of the acylated insulin analogue per ml of solution. The formulation can include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and, preferably, zinc. The formulation can also include an excipient or agent for stabilization of the acylated insulin analogue, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins useful in formulating insulin conjugates include albumin, protamine, or the like. Typical carbohydrates useful in formulating the acylated insulin analogue include sucrose, mannitol, lactose, trehalose, glucose; or the like. The acylated insulin analogues formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the insulin conjugate caused by atomization of the solution in forming an aerosol. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between about 0.001 and about 4% by weight of the formulation.

Pharmaceutical compositions containing an acylated insulin analogue of this invention may also be administered parenterally to patients in need of such a treatment. Parenteral administration may be performed by subcutaneous, intramuscular or intravenous injection by means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump.

Injectable compositions of the acylated insulin analogues of this invention can be prepared using the conventional techniques of the pharmaceutical industry which involve dissolving and mixing the ingredients as appropriate to give the desired end product. Thus, according to one procedure, an acylated insulin analogue is dissolved in an amount of water which is somewhat less than the final volume of the composition to be prepared. Zink, an isotonic agent, a preservative and/or a buffer is/are added as required and the pH value of the solution is adjusted - if necessary - using an acid, e.g., hydrochloric acid, or a base, e.g., aqueous sodium hydroxide as needed. Finally, the volume of the solution is adjusted with water to give the desired concentration of the ingredients.

In a further embodiment of this invention the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each one of these specific buffers constitutes an alternative embodiment of this invention.

In a further embodiment of this invention the formulation further comprises a pharmaceutically acceptable preservative which may be selected from the group consisting of phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethonium chloride, chlorphenesine (3-(4-chlorophenoxy)-1,2-propanediol) or mixtures thereof. In a further embodiment of this invention the preservative is present in a concentration from about 0.1 mg/ml to 20 mg/ml. In a further embodiment of this invention the preservative is present in a concentration from about 0.1 mg/ml to 5 mg/ml. In a further embodiment of this invention the preservative is present in a concentration from about 5 mg/ml to 10 mg/ml. In a further embodiment of this invention the preservative is present in a concentration from about 10 mg/ml to 20 mg/ml. Each one of these specific preservatives constitutes an alternative embodiment of this invention. The use of a preservative in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19^(th) edition, 1995.

In a further embodiment of this invention, the formulation further comprises an isotonic agent which may be selected from the group consisting of a salt (e.g., sodium chloride), a sugar or sugar alcohol, an amino acid (for example, L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or threonine), an alditol (e.g. glycerol (glycerine), 1,2-propanediol (propyleneglycol), 1,3-propanediol or 1,3-butanediol), polyethyleneglycol (e.g., PEG400) or mixtures thereof. Any sugar such as mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na may be used. In one embodiment the sugar additive is sucrose. Sugar alcohol is defined as a C4-C8 hydrocarbon having at least one —OH group and includes, e.g., mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. In one embodiment the sugar alcohol additive is mannitol. The sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, as long as the sugar or sugar alcohol is soluble in the liquid preparation and does not adversely effect the stabilizing effects achieved using the methods of this invention. In one embodiment, the sugar or sugar alcohol concentration is between about 1 mg/ml and about 150 mg/ml. In a further embodiment of this invention the isotonic agent is present in a concentration from about 1 mg/ml to 50 mg/ml. In a further embodiment of this invention the isotonic agent is present in a concentration from about 1 mg/ml to 7 mg/ml. In a further embodiment of this invention the isotonic agent is present in a concentration from about 8 mg/ml to 24 mg/ml. In a further embodiment of this invention the isotonic agent is present in a concentration from about 25 mg/ml to 50 mg/ml. Each one of these specific isotonic agents constitutes an alternative embodiment of this invention. The use of an isotonic agent in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19^(th) edition, 1995.

Typical isotonic agents are sodium chloride, mannitol, dimethyl sulfone and glycerol and typical preservatives are phenol, m-cresol, methyl p-hydroxybenzoate and benzyl alcohol.

Examples of suitable buffers are sodium acetate, glycylglycine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and sodium phosphate.

A composition for nasal administration of an acylated insulin analogues of this invention may, e.g., be prepared as described in European Patent No. 272097.

Compositions containing acylated insulin analogues of this invention can be used in the treatment of states which are sensitive to insulin. Thus, they can be used in the treatment of type 1 diabetes, type 2 diabetes and hyperglycaemia for example as sometimes seen in seriously injured persons and persons who have undergone major surgery. The optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific insulin derivative employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the state to be treated. It is recommended that the daily dosage of the acylated insulin analogue of this invention be determined for each individual patient by those skilled in the art in a similar way as for known insulin compositions.

Important Features of the Invention

To sum up, some features of this invention are as follows:

1. An acylated insulin analogue wherein the insulin analogue comprises a lysine residue connected C-terminally to the A21 amino acid residue or a peptide residue of up to 4 amino acid residues comprising a lysine residue which peptide residue is connected C-terminally to the A21 amino acid residue, characterized in that an acyl moiety comprising an alkylene glycol moiety is attached to the lysine residue in the A22 position or attached to a lysine residue present in the peptide residue that is attached to the C terminal end of the A21 amino acid residue and wherein there is only one lysine (K, Lys) in the insulin analogue.

2. An acylated insulin analogue, according to clause 1, wherein the acyl moiety has the general formula I:

Acy-AA1_(n)-AA2_(m)-AA3_(p)-  (I),

wherein

n is 0 or an integer in the range 1-3,

m is 0 or an integer in the range 1-6,

p is1,2 or 3,

Acy is a fatty acid or a fatty diacid comprising about 8-24 carbon atoms from which, formally, a hydroxy group has been removed from the carboxy group of the fatty acid or from one of the carboxy groups of the fatty diacid,

AA1 is a neutral cyclic amino acid from which, formally, a hydrogen atom has been removed from the amino group and a hydroxy group has been removed from the carboxy group,

AA2 is an acidic amino acid from which, formally, a hydrogen atom has been removed from the amino group and a hydroxy group has been removed from the carboxy group,

AA3 is a neutral, alkyleneglycol-containing amino acid from which, formally, a hydrogen atom has been removed from the amino group and a hydroxy group has been removed from the carboxy group,

the order by which AA1, AA2 and AA3 appears in the formula can be interchanged independently,

the connections between Acy, AA1, AA2 and/or AA3 are amide (peptide) bonds, and

attachment to the parent insulin can be from the C-terminal end of a AA1, AA2, or AA3 residue in the acyl moiety of the formula (I) or from one of the side-chain(s) of an AA2 residue present in the moiety of formula (I).

3. An acylated insulin analogue wherein the insulin analogue comprises a lysine residue connected C-terminally to the A21 amino acid residue or a peptide residue of up to 4 amino acid residues comprising a lysine residue which peptide residue is connected C-terminally to the A21 amino acid residue, characterized in that an acyl moiety comprising an alkylene glycol moiety is attached to the lysine residue in the A22 position or attached to a lysine residue present in the peptide residue that is attached to the C terminal end of the A21 amino acid residue and wherein there is only one lysine (K, Lys) in the insulin analogue and wherein the acyl moiety comprising an alkylene glycol moiety has the general formula I: Acy-AA1_(n)-AA2_(m)-AA3_(p)- (I), wherein n is 0 or an integer in the range 1-3, m is 0 or an integer in the range 1-6, p is 1, 2 or 3, Acy is a fatty acid or a fatty diacid comprising about 8-24 carbon atoms from which, formally, a hydroxy group has been removed from the carboxy group or from one of the carboxy groups, AA1 is a neutral cyclic amino acid from which, formally, a hydrogen atom has been removed from the amino group and a hydroxy group has been removed from the carboxy group, AA2 is an acidic amino acid from which, formally, a hydrogen atom has been removed from the amino group and a hydroxy group has been removed from the carboxy group, AA3 is a neutral, alkyleneglycol-containing amino acid from which, formally, a hydrogen atom has been removed from the amino group and a hydroxy group has been removed from the carboxy group, the order by which AA1, AA2 and AA3 appears in the formula can be interchanged independently, the connections between Acy, AA1, AA2 and/or AA3 are amide (peptide) bonds, and attachment to the parent insulin can be from the C-terminal end of a AA1, AA2, or AA3 residue in the acyl moiety of the formula (I) or from one of the side-chain(s) of an AA2 residue present in the moiety of formula (I).

4. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein Acy originates from a fatty acid or a fatty diacid with 14 to 20 carbon atoms, preferably 16 to 20 carbon atoms, more preferred 16 to 18 carbon atoms, alternatively 18 to 20 carbon atoms and specifically 8, 10, 12, 14, 16, 17, 18, 20, 22 or 24 carbon atoms.

5. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein Acy originates from a dicarboxylic acid.

6. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein Acy is tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid, heptadecanedioic acid, octadecanedioic acid, or eicosanedioic acid from which a hydroxy group has been removed.

7. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein Acy is hexadecanedioic acid, octadecanedioic acid, or eicosanedioic acid from which a hydroxy group has been removed.

8. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein AA1 is selected from:

wherein q is 0, 1, 2, 3 or 4, and from which, formally, a hydrogen atom has been removed from the amino group and a hydroxy group has been removed from the carboxy group.

9. An acylated insulin analogue, according to the preceding clause, wherein AA1 is

from which, formally, a hydrogen atom has been removed from the amino group and a hydroxy group has been removed from the carboxy group.

10. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein AA2 is selected from Glu, Asp, D-Glu, D-Asp, γGlu, γAsp, γ-D-Glu, γ-D-Asp, or any of the following compounds:

wherein the arrows indicate the attachment point to the amino group of AA1, AA2 or AA3 and from which, formally, a hydrogen atom has been removed from the amino group and a hydroxy group has been removed from the carboxy group.

11. An acylated insulin analogue, according to the preceding clauses, wherein AA2 is γGlu.

12. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein AA3 is selected from any of the following compounds:

r is an integer in the range 1-100, preferably 1-10,

s is an integer in the range 1-30, preferably 1-10,

t is an integer in the range 1-150, preferably 20-70,

and from which, formally, a hydrogen atom has been removed from the amino group and a hydroxy group has been removed from the carboxy group.

13. An acylated insulin analogue, according to the preceding clause, wherein r is 1, 2, 3, 4, 5, 7, 11, 23 or 27.

14. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein s is 1, 2, 3, 4, 10, 20 or 30.

15. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the integer t is selected so that the average molecular weight of the above formula wherein the integer t appears is 2000 Da, 3400 Da or 5000 Da.

16. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the acyl moiety of the formula Acy-AA1_(n)-AA2_(m)-AA3_(p)-, wherein Acy, AA1, AA2, AA3, n, m and p are as defined above, is connected to a lysine residue in position A22.

17. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the acyl moiety of the formula Acy-AA1_(n)-AA2_(m)-AA3,-, wherein Acy, AA1, AA2, AA3, n, m and p are as defined above, is connected to a lysine residue in position A23.

18. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the acyl moiety of the formula Acy-AA1_(n)-AA2_(m)-AA3_(p)-, wherein Acy, AA1, AA2, AA3, n, m and p are as defined above, is connected to a lysine residue in position A24.

19. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the acyl moiety of the formula Acy-AA1_(n)-AA2_(m)-AA3_(p)-, wherein Acy, AA1, AA2, AA3, n, m and p are as defined above, is connected to a lysine residue in position A25.

20. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the insulin analogue comprises 52 amino acid residues.

21. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the insulin analogue comprises 51 amino acid residues.

22. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the insulin analogue comprises 53 amino acid residues.

23. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the insulin analogue comprises 54 amino acid residues.

24. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the insulin analogue comprises 50 amino acid residues.

25. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the insulin analogue comprises 49 amino acid residues.

26. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein only one of the amino acid residues in the positions A1-A21 and B1-B30 of the insulin analogue deviates from those amino acid residues present in human insulin.

27. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein only two of the amino acid residues in the positions A1-A21 and B1-B30 of the insulin analogue deviates from those amino acid residues present in human insulin.

28. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein only three of the amino acid residues in the positions A1-A21 and B1-B30 of the insulin analogue deviates from those amino acid residues present in human insulin.

29. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein only four of the amino acid residues in the positions A1-A21 and B1-B30 of the insulin analogue deviates from those amino acid residues present in human insulin.

30. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein there is no amino acid residue connected to the N terminal end of the amino acid residues present in position A1 or B1 and no amino acid residue connected to the C terminal end of the amino acid residues present in position B30.

31. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the amino acid residue in the A14 position of the insulin analogue is E.

32. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the amino acid residue in the A18 position of the insulin analogue is Q.

33. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the amino acid residue in the A21 position of the insulin analogue is A, G or Q.

34. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the amino acid residue in the A22 position of the insulin analogue is K or G.

35. An acylated insulin analogue, according to the preceding clause, wherein the amino acid residue in the A22 position of the insulin analogue is K.

36. An acylated insulin analogue, according to the preceding clause, wherein the amino acid residue in the A22 position of the insulin analogue is K and wherein there is no amino acid residue connected to the C terminal end of said A22K amino acid residue.

37. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the amino acid residue in the A23 position of the insulin analogue is K or G.

38. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the amino acid residue in the A24 position of the insulin analogue is K or G.

39. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the amino acid residue in the A25 position of the insulin analogue is K.

40. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the amino acid residue in the B1 position of the insulin analogue is Q or absent.

41. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the amino acid residue in the B3 position of the insulin analogue is Q or T.

42. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the amino acid residue in the B13 position of the insulin analogue is Q.

43. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the amino acid residue in the B25 position of the insulin analogue is H.

44. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the amino acid residue in the B27 position of the insulin analogue is absent.

45. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the amino acid residue in the B28 position of the insulin analogue is D, E or R.

46. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the amino acid residue in the B28 position is R and the amino acid residue in the B29 position is P.

47. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the amino acid residue in the B29 position of the insulin analogue is R.

48. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the amino acid residue in the B30 position of the insulin analogue is absent.

49. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the amino acid residue in the A14 position of the insulin analogue is Y or E, the amino acid residue in the Alb position of the insulin analogue is N or Q, the amino acid residue in the A21 position of the insulin analogue is N, A, G or Q, the amino acid residue in the A22 position of the insulin analogue is K or G, the amino acid residue in the A23 position of the insulin analogue is absent, K or G, the amino acid residue in the A24 position of the insulin analogue is absent, K or G, the amino acid residue in the A25 position of the insulin analogue is absent or K, the amino acid residue in the B1 position of the insulin analogue is F, Q or absent, the amino acid residue in the B3 position of the insulin analogue is N, Q or T, the amino acid residue in the B13 position of the insulin analogue is E or Q, the amino acid residue in the B25 position of the insulin analogue is F or H, the amino acid residue in the B27 position of the insulin analogue is T or absent, the amino acid residue in the B28 position of the insulin analogue is P, D, E or R, the amino acid residue in the B29 position of the insulin analogue is K or R and the amino acid residue in the B30 position of the insulin analogue is T or absent.

50. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein all the amino acid residues in the insulin analogue are residues of codable amino acids.

51. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein AA2 is gGlu.

52. An acylated insulin analogue, according to any one of the preceding, possible clauses, wherein the the acyl moiety of the general formula Acy-AA1_(n)-AA2_(m)-AA3_(p)- has one of the general formulae Acy-AA3-, Acy-AA2-AA3-, Acy-AA2-AA3-AA2-, Acy-AA2-(AA3)₂-, Acy-AA2-(AA3)₂-AA2 or Acy-M3-AA2- wherein Acy, M2 and AA3 each is as defined herein.

53. An acylated insulin analogue, according to any one of the preceding, possible clauses, which is any one of the compounds mentioned specifically herein, e.g., the compounds described in the specific examples herein.

54. A compound according to any one of the preceding, possible product clauses for use as a medicament or for use in a medicament.

55. A compound according to any one of the preceding, possible product clauses for treating diabetes or the use of a compound according to any one of the preceding, possible product claims for the preparation of a medicament for the treatment of diabetes.

56. A method for treatment of diabetes, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to any one of the preceding, possible product clauses.

57. Any novel feature or combination of features described herein.

Combining one or more of the embodiments described herein, optionally also with one or more of the claims below and one or more of the clauses mentioned herein, results in further embodiments and the present invention relates to all possible combinations of said embodiments, clauses and claims.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law).

All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents. The mentioning herein of references is no admission that they constitute prior art.

Herein, the word “comprise” is to be interpreted broadly meaning “include”, “contain” or “comprehend” (EPO guidelines C 4.13).

This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.

The following examples are offered by way of illustration, not by limitation.

EXAMPLES General Procedures

Construction of Expression Vectors, Transformation of the Yeast Cells, and Expression of the Insulin Precursors of the Invention

All expressions plasmids are of the C-POT type, similar to those described in EP 171142, which are characterized by containing the Schizosaccharomyces pombe triose phosphate isomerase gene (POT) for the purpose of plasmid selection and stabilization in S. cerevisiae. The plasmids also contain the S. cerevisiae triose phosphate isomerase promoter and terminator. These sequences are similar to the corresponding sequences in plasmid pKFN1003 (described in WO 90/10075) as are all sequences except the sequence of the EcoRI-XbaI fragment encoding the fusion protein of the leader and the insulin product. In order to express different fusion proteins, the EcoRI-XbaI fragment of pKFN1003 is simply replaced by an EcoRI-XbaI fragment encoding the leader-insulin fusion of interest. Such EcoRI-XbaI fragments may be synthesized using synthetic oligonucleotides and PCR according to standard techniques.

Yeast transformants were prepared by transformation of the host strain S. cerevisiae strain MT663 (MATalMATα pep4-3/pep4-3 HIS4/his4 tpi::LEU2/tpi::LEU2 Cir⁺). The yeast strain MT663 was deposited in the Deutsche Sammlung von Mikroorganismen and Zellkulturen in connection with filing WO 92/11378 and was given the deposit number DSM 6278.

MT663 was grown on YPGaL (1% Bacto yeast extract, 2% Bacto peptone, 2% galactose, 1% lactate) to an O.D. at 600 nm of 0.6. 100 ml of culture was harvested by centrifugation, washed with 10 ml of water, recentrifuged and resuspended in 10 ml of a solution containing 1.2 M sorbitol, 25 mM Na₂EDTA pH=8.0 and 6.7 mg/ml dithiotreitol. The suspension was incubated at 30° C. for 15 minutes, centrifuged and the cells resuspended in 10 ml of a solution containing 1.2 M sorbitol, 10 mM Na₂EDTA, 0.1 M sodium citrate, pH 0 5.8, and 2 mg Novozym® 234. The suspension was incubated at 30° C. for 30 minutes, the cells collected by centrifugation, washed in 10 ml of 1.2 M sorbitol and 10 ml of CAS (1.2 M sorbitol, 10 mM CaCl₂, 10 mM Tris HCl (pH=7.5) and resuspended in 2 ml of CAS. For transformation, 1 ml of CAS-suspended cells was mixed with approx. 0.1 mg of plasmid DNA and left at room temperature for 15 minutes. 1 ml of (20% polyethylene glycol 4000, 10 mM CaCl₂, 10 mM Tris HCl, pH=7.5) was added and the mixture left for a further 30 minutes at room temperature. The mixture was centrifuged and the pellet resuspended in 0.1 ml of SOS (1.2 M sorbitol, 33% v/v YPD, 6.7 mM CaCl₂) and incubated at 30° C. for 2 hours. The suspension was then centrifuged and the pellet resuspended in 0.5 ml of 1.2 M sorbitol. Then, 6 ml of top agar (the SC medium of Sherman et al. (1982) Methods in Yeast Genetics, Cold Spring Harbor Laboratory) containing 1.2 M sorbitol plus 2.5% agar) at 52° C. was added and the suspension poured on top of plates containing the same agar-solidified, sorbitol containing medium. S. cerevisiae strain MT663 transformed with expression plasmids was grown in YPD for 72 h at 30° C.

Production, Purification and Characterization of the Acylated Insulin Analogues of this Invention

The following examples refer to intermediate compounds and final products identified in the specification and in the examples. The preparation of the acylated insulin analogues of this invention is described in detail using the following examples, but the chemical reactions and purification schemes described are disclosed in terms of their general applicability to the preparation of the insulin derivatives of the invention. Occasionally, the reaction may not be applicable as described to each compound included within the disclosed scope of the invention. The compounds for which this occurs will be readily recognised by those skilled in the art. In these cases, the reactions can be successfully performed by conventional modifications known to those skilled in the art, that is, by appropriate protection of interfering groups, by changing to other conventional reagents, or by routine modification of reaction conditions. Alternatively, other reactions disclosed herein or otherwise conventional will be applicable to the preparation of the corresponding compounds of the invention. In all preparative methods, all starting materials are known or may easily be prepared from known starting materials using methods known per se. All temperatures are set forth in degrees Celsius and, unless otherwise indicated, all parts and percentages are by weight when referring to yields and all parts are by volume when referring to solvents and eluents.

The acylated insulin analogues of this invention can be purified by employing one or more of the following procedures which are typical within the art. These procedures can—if needed—be modified with regard to gradients, pH, salts, concentrations, flow, columns and so forth. Depending on factors such as impurity profile, solubility of the insulins in question etcetera, these modifications can readily be recognised and made by a person skilled in the art.

General Procedure for the Solid Phase Synthesis of Acylation Reagents of the General Formula (II):

Acy-AA1_(n)-AA2_(m)-AA3_(p)-Act,  (II):

wherein Acy, AA1, AA2, AA3, n, m, and p are as defined above and Act is the leaving group of an active ester, such as N-hydroxysuccinimide (OSu), or 1-hydroxybenzotriazole, esters and wherein carboxylic acids within the acyl moiety are protected as tort-butyl esters.

Compounds of the general formula (II) according to the invention can be synthesised on solid support using procedures well known to skilled persons in the art of solid phase peptide synthesis. This procedure comprises attachment of a Fmoc protected amino acid to a polystyrene 2-chlorotritylchloride resin. The attachment can, e.g., be accomplished using the free N-protected amino add in the presence of a tertiary amine, like triethyl amine or N,N-diisopropylethylamine (see references below). The C-terminal end (which is attached to the resin) of this amino acid is at the end of the synthetic sequence being coupled to the parent insulins of the invention. After attachment of the Fmoc amino acid to the resin, the Fmoc group is deprotected using, e.g., secondary amines, like piperidine or diethyl amine, followed by coupling of another (or the same) Fmoc protected amino acid and deprotection. The synthetic sequence is terminated by coupling of mono-tert-butyl protected fatty (α, ω) diacids, like hexadecanedioic, heptadecanedioic, octadecanedioic or eicosanedioic acid mono-tert-butyl esters. Cleavage of the compounds from the resin is accomplished using diluted acid like 0.5-5% TFA/DCM (trifluoroacetic acid in dichloromethane), acetic acid (eg. 10% in DCM, or HOAc/triflouroethanol/DCM 1:1:8), or hecafluoroisopropanol in DCM (See eg. “Organic Synthesis on Solid Phase”, F. Z. Dörwald, Wiley-VCH, 2000. ISBN 3-527-29950-5, “Peptides: Chemistry and Biology”, N. Sewald & H.-D. Jakubke, Wiley-VCH, 2002, ISBN 3-527-30405-3 or “The Combinatorial Cheemistry Catalog” 1999, Novabiochem AG, and references cited therein). This ensures that tert-butyl esters present in the compounds as carboxylic acid protecting groups are not deprotected. Finally, the C-terminal carboxy group (liberated from the resin) is activated, eg. as the N-hydroxysuccinimide ester (OSu) and used either directly or after purification as coupling reagent in attachment to parent insulins of the invention. This procedure is illustrated in example 4.

Alternatively, the acylation reagents of the general formula (II) above can be prepared by solution phase synthesis as described below.

Mono-tert-butyl protected fatty diacids, such as hexadecanedioic, heptadecanedioic, octadecanedioic or eicosanedioic acid mono-cert-butyl esters are activated eg. as OSu-esters as described below or as any other activated ester known to those skilled in the art, such as HOBt- or HOAt-esters. This active ester is coupled with one of the amino acids AA1, mono-tert-butyl protected AA2, or AA3 in a suitable solvent such as THF, DMF, NMP (or a solvent mixture) in the presence of a suitable base, such as DIPEA or triethylamine. The intermediate is isolated, eg by extractive procedures or by chromatographic procedures. The resulting intermediate is again subjected to activation (as described above) and to coupling with one of the amino acids AA1, mono-tort-butyl protected AA2, or AA3 as described above. This procedure is repeated until the desired protected intermediate Acy-AA1_(n)-AA2_(m)-AA3_(p)-OH is obtained. This is in turn activated to afford the acylation reagents of the general formula (II) Acy-AA1_(n)-AA2_(m)-AA3_(p)-Act. This procedure is illustrated in example.

General Procedure (A) for Preparation of Acylated Insulin Analogues of this Invention

The general procedure (A) is outlined below and illustrated in the first examples:

Example 1 General Procedure (A)

A22K(N^(ε)-Hexadecandioyl-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl-γGlu), B29R, desB30 human Insulin

This insulin was prepared in analogy with the insulin described in WO 2006/082205, example 3, using A22K, B29R desB30 human insulin instead of desB30 human insulin.

LC-MS (electrospray); m/z: 1627 (M+4)⁺/4; 1302 (M+5)⁺/5.

Example 2 General Procedure (A)

A22K(N^(ε)-Hexadecandioyl-(2-aminoethyl-PEG2000-ylacetyl)), B29R desB30 human insulin

Step 1:

Hexadecanedioic acid tert-butyl ester 2,5-dioxopyrrolidin-1-yl ester (218 mg, 0.497 mmol) was dissolved in DCM (10 mL), propylamine-PEG-carboxylic acid MW 2000 (SUNBRIGHT® PA-20HC from NOF coporation, 1000 mg, approximately 0.497 mmol) was added and the mixture was stirred under nitrogen overnight. The reaction mixture was transferred to a silica gel column and eluted with DCM with a gradient of ethanol (0-10%), to obtained the tert-butylhexadecandioylpropylamine-PEG-carboxylic acid (MW 2000) intermediate.

Step 2: Synthesis of A22K(N^(ε)-Hexadecandioyl-(2-aminoethyl-PEG2000-ylacetyl)), B29R, desB30 human insulin.

tert-Butylhexadecandioylpropylamine-PEG-carboxylic acid (MW 2000) (72 mg, 0.031 mmol) was dissolved in THF (4 mL), DIPEA (6.3 μL, 0.037 mmol) and TSTU (11.1 mg, 0.037 mmol) was added and the mixture was stirred overnight under nitrogen, DMSO (1 mL) was added and the mixture was added to a solution of A22K, B29R desB30 human insulin (180 mg, 0.031 mmol) dissolved together with triethylamin (43 μL, 0.31 mmol) in DMSO (2 mL). The mixture was cooled on ice and added water (15 mL), pH was fist adjusted to 5.2 with 1N HCl and then adjusted back to pH 7-8 with 1% ammonia in water. The mixture was purified on preparative HPLC using a 35-75% gradient of acetonitrile in water containing 0.1% TFA. Fractions were analyzed on LC-MS and fractions containing the desired product were pooled, solvent removed in vacuo followed by lyophilisation. The lyophilized material was treated with TFA/water 95:5 for 30 minutes and the solvent was removed in vacuo. The crude product was purified using anion exchange chromatography (Ressource Q column, 1 ml (Amersham Biosciences) and elution with a gradient of ammonium acetate (0.25% to 1.25% over 25 CV) in 42.5% EtOH, 0.25% TRIS, pH 7.5, followed by preparative HPLC using a Jupiter C4, 5μ column eluted with a 30-70% acetonitrile/water gradient containing 0.1% TFA over 90 minutes to give the title compound.

HPLC-MS: m/z around 1360 (M⁺/6).

Example 3 General Procedure (A)

A22K(N^(ε)-3-(3-{4-[3-(5-Carboxypentanoylamino)propoxy]butoxy}propylcarbamoyl)propionyl-γGlu), B29R, desB30 human insulin

This insulin was prepared in analogy with the insulin described in WO 2006/082205, example 8, using A22K, B29R desB30 human insulin instead of desB30 human insulin and using hexanedioic acid mono-tert-butyl ester instead of octanedioic acid mono-tert-butyl ester.

MALDI-TOF MS (matrix: sinapinic acid); m/z: 6411.

Example 4 General Procedure (A)

A22K(N^(ε)-[2-(2-[2-(2-[2-(Octadecandioyl-γGlu)amino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]), B29R, desB30 human insulin

MALDI-TOF MS (matrix: sinapinic acid); m/z: 6581.

The acylation reagent for preparation of this insulin was prepared as described in the following:

Starting resin: 2-Chlorotrityl resin, 1.60 mmol/g 1.0 g of the resin was swelled for 30 min in DCM (10 ml).

1. Acylation with Fmoc-8-amino-3 6-dioxaoctanoic acid:

0.39 g (0.63 eq, 1.0 mmol) of Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-OEG-OH) was dissolved in DCM (15 ml) and was added to the resin. N,N-Diisopropylethylamine (DIEA) (0.44 ml, 2.5 mmol) was added dropwise. The reaction mixture was vortexed for 30 min. and then methanol (2 ml) was added and the mixture was vortexed for additional 15 min. The resin was filtered and washed with NMP (2×8 ml) and DCM (8×8 ml).

20% piperidine/NMP (8 ml) was added, standing 10 min. repeated once.

Filtered and washed with NMP (2×8 ml), DCM (3×8 ml), and NMP (5×8 ml).

A positive TNBS test gave red-coloured resins.

2. Acylation with Fmoc-8-amino-3,6-dioxaoctanoic acid:

0.78 g (2 eq, 2.0 mmol) of Fmoc-8-amino-3,6-dioxaoctanoic acid was dissolved in NMP/DCM 1:1 (10 ml). 0.28 g (2.2 eq, 2.4 mmol) of HOSu was added followed by addition of 0.37 ml (2.2 eq, 2.4 mmol) of DIC. The reaction mixture was allowed to stand for 1 hour and was then added to the resin and finally 0.407 ml (2.2 eq) of DIEA was added. The mixture was vortexed for 16 hours, filtered and washed with NMP (2×8 ml), DCM (3×8 ml), and NMP (5×8 ml).

A positive TNBS test gave colourless resins.

20% piperidine/NMP (10 ml) was added, standing 10 min. repeated once.

Filtered and washed with NMP (2×8 ml), DCM (3×8 ml), and NMP (5×8 ml).

A positive TNBS test gave red-coloured resins.

Acylation with Fmoc-Glu-OtBu:

0.86 g (2 eq, 2.0 mmol) of Fmoc-Glu-OtBu was dissolved in NMP/DCM 1:1 (10 ml). 0.32g (2.2 eq, 2.4 mmol) of HOBT was added followed by addition of 0.37 ml (2.2 eq, 2.4 mmol) of DIC. The reaction mixture was allowed to stand for 20 min and was then transferred to the resin and finally 0.407 ml (2.2 eq) of DIEA was added. The mixture was vortexed for 16 hours, filtered and washed with NMP (2×8 ml), DCM (3×8 ml), and NMP (5×8 ml).

A positive TNBS test gave colourless resins.

20% piperidine/NMP (10 ml) was added, standing 10 min. repeated once.

Filtered and washed with NMP (2×8 ml), DCM (3×8 ml), and NMP (5×8 ml).

A positive TNBS test gave red-coloured resins.

Acylation with Octadecanedioic Acid Mono tert-butyl ester:

0.75 g (2 eq, 2.0 mmol) Octadecanedioic acid mono tert-butyl ester was dissolved NMP/DCM 1:1 (10 ml). 0.32 g (2.2 eq, 2.4 mmol) HOBT was added followed by addition of 0.37 ml (2.2 eq, 2.4 mmol) of DIC. The reaction mixture was allowed to stand for 20 min and was then transferred to the resin and finally 0.41 ml (2.2 eq) of DIEA was added. The mixture was vortexed for 16 hours, filtered and washed with NMP (2×8 ml), DCM (3×8 ml), and NMP (5×8 ml).

Cleavage with TFA:

8 ml of 5% TFA/DCM was added to the resin and the reaction mixture was vortexed for 2 hours, filtered and the filtrate was collected. More 5% TFA/DCM (8 ml) was added to the resin, and the mixture was vortexed for 10 min, filtered and the resin was washed with DCM (2×10 ml). The combined filtrates and washings were pH adjusted to basic using about 800 ul of DIEA. The mixture was evaporated in vacuo affording an oil (3.5 g). Diethylether (30 ml) was added and the not dissolved oil was separated by decantation and evaporated in vacuo. This afforded 1.1 g of 17-{(S)-1-tert-butoxy-carbonyl-3-[2-(2-{[2-(2-carboxymethoxyethoxy)ethylcarbamoyl]methoxy}ethoxy)ethylcarbamoyl]propyl-carbamoyl}heptadecanoic acid tert-butyl ester (alternative name: Tert-butyl octadecandioyl-Glu(OEG-OEG-OH)-OTBU) as an oil.

LC-MS (Sciex100 API): m/z=846.6 (M+1)⁺.

OSu-Activation:

The above tert-butyloctadecandioyl-Glu(OEG-OEG-OH)-OTBU (0.63 g) was dissolved in THF (35 ml). DIEA (0.255 ml, 2 eq.) was added followed by TSTU (0.45 g, 2 eq.), and the mixture was stirred at room temperature for 16 hours. The mixture was partitioned between ethyl acetate (250 ml) and aqueous NaHSO₄ (3×100 ml). The organic phase was dried (MgSO₄) and concentrated in vacuo to afford 0.65 g of 17-((S)-1-tert-butoxycarbonyl-3-{2-[2-({2-[2-(2,5-dioxopyrrolidin-1-yloxycarbonylmethoxy)-ethoxy]ethylcarbamoyl}methoxy)ethoxy]ethylcarbamoyl}propylcarbamoyl)heptadecanoic acid tert-butyl ester (alternative name: tert-butyl octadecandioyl-Glu(OEG-OEG-OSu)-oTBU) as an oil.

LC-MS: m/z=943.4 (M+1)⁺.

Example 5 General Procedure (A)

A22K(N^(ε)-3-(3-{4-[3-(13-Carboxytridecanoylamino)propoxy]butoxy}propylcarbamoyl)propionyl-γGlu), B29R, desB30 human insulin

LC-MS (electrospray):=1630, (M+4)⁺/4

Example 6 General Procedure (A)

A22K(N^(ε)-[2-(2-[2-(2-[2-(Eicosanedioyl-γGlu)amino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]), B29R, desB30 human insulin

MALDI-MS (matrix: sinapinic acid); m/z: 6606

Example 7 General Procedure (A)

A14E, A22K(N^(ε)-[2-(2-[2-(2-[2-(Octadecandioyl-γGlu)amino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)-acetyl]), B25H, B29R, desB30 human insulin

LC-MS (electrospray): m/z=1634, (M+4)⁺/4

Example 8 General Procedure (A)

A22K(N^(ε)-Octadecandioyl-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), desB29, desB30 human insulin

63 mg A22K(N^(ε)-octadecandioyl-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]-acetyl), B29R, desB30 human insulin (example 4) was dissolved in Tris buffer, pH 8 (50 mM, 10 mL), and Carboxypeptidase B immobilised on sepharose gel in Tris buffer, pH 8 (50 mM, 0.2 mL) containing 20% ethanol was added and the mixture was gently shaken for 16 hours at room temperature. More Carboxypeptidase B immobilised on sepharose gel in Tris buffer, pH 8 (50 mM, 0.8 mL) containing 20% ethanol was added and the mixture was gently shaken for 24 hours. The mixture was filtered and the filter was washed with Tris buffer, pH 8 (50 mM, 0.8 mL) containing 20% ethanol. The combined filtrates and washings were lyophilised. The residue was purified by HPLC to afford the title insulin

MALDI-TOF MS: m/z=6424

Example 9 General Procedure (A)

A14E, A22K(N^(ε)-Eicosanedioyl-γGlu-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl)), B25H, B29R, desB30 human insulin

Preparation of the acylation reagent 19-{1-tert-Butoxycarbonyl-3-[2-(2-{2[2-(2-carboxyethoxy)ethoxy]-ethoxy}ethoxy)ethylcarbamoyl]propylcarbamoyl}nonadecanoic acid tert-butyl ester N-hydroxy-succinimide ester and coupling to the parent insulin:

Icosanedioic acid tert-butyl ester N-hydroxysuccinimide ester:

Eicosanedioic acid mono-tert-butyl ester (5 g, 12.54 mmol) and TSTU (4.53 g, 15.05 mmol) were mixed in THF (50 ML), DIPEA (2.62 mL) was added and the resulting cloudy mixture was stirred at RT for 2 h, then DMF (30 mL) was added resulting in a clear solution which was further stirred overnight. The resulting mixture was evaporated to almost dryness and the residue was mixed with cold acetonitrile resulting in the precipitation of a precipitate. This was filtered off and dried in vacuum overnight, affording 6.01 g (97%) of icosanedioic acid tert-butyl ester N-hydroxysuccinimide ester.

MS (electrospray): m/z: 440 (M−56 (tBu)).

2-(19-tert-Butoxycarbonylnonadecanoylamino)pentanedioic acid 1-tert-butyl ester

Icosanedioic acid tert-butyl ester 2,5-dioxo-pyrrolidin-1-yl ester (6.01 g, 12.124 mmol) was dissolved in THF (150 mL) and mixed with a slurry of H-Glu-OtBu (2.71 g, 13.33 mmol) in DMF/water (1/1, 40 mL). This resulted in a gel-like solution which was heated to give a clear solution that was stirred at RT for 3 h. Then the solution was evaporated, 100 mL of water was added and the mixture was heated to 60° C. which resulted in a solution which crystallised on cooling. The precipitate was recrystallised from acetonitrile and the crystals were dried in vacuum. Yield 6.82 g (96%).

MS (electrospray): m/z 584 (M+1).

2-(19-tert-Butoxycarbonylnonadecanoylamino)pentanedioic acid 1-tert-butyl ester 5-(2,5-dioxopyrrolidin-1-yl) ester

2-(19-tert-Butoxycarbonylnonadecanoylamino)pentanedioic acid 1-tert-butyl ester (6.52 g, 11.17 mmol) was dissolved in THF (100 mL), DIPEA (2.14 mL) was added followed by a solution of TSTU (3.70 g, 12.29 mmol) in acetonitrile (25 mL). The mixture was stirred overnight at RT, then it was evaporated resulting in a brownish residue which was recrystallised from acetonitrile. After cooling overnight at 5° C. a powder was formed. This was dissolved in THF and dried with MgSO₄, filtered and evaporated to dryness to afford 6.17 g (81%) of the title compound.

MS (electrospray): m/z: 681 (M+1).

19-{1-tert-Butoxycarbonyl-3-[2-(2-{2-[2-(2-carboxyethoxy)ethoxy]ethoxy}ethoxy)ethylcarbamoyl]propyl-carbamoyl}nonadecanoic acid tert-butyl ester

2-(19-tert-Butoxycarbonylnonadecanoylamino)pentanedioic acid 1-tert-butyl ester 5-(2,5-dioxopyrrolidin-1-yl) ester (0.56 g, 0.822 mmol) was dissolved in THF (20 mL), 3-(2-{2-[2-(2-amino-ethoxy)-ethoxy]ethoxy}ethoxy)propionic acid (0.22 g, 0.82 mmol) in THF (20 mL) was added and the mixture stirred at RT for 3 h. LCMS indicated the reaction was finished (MS: m/z 831 (M+1)) and the reaction mixture was used directly for the following reaction with TSTU.

19-(1-tert-Butoxycarbonyl-3-{2-[2-(2-{2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonyl)ethoxy]ethoxy}ethoxy)ethoxy]ethylcarbamoyl}propylcarbamoyl)nonadecanoic acid tert-butyl ester

19-{1-tert-Butoxycarbonyl-3-[2-(2-{2-[2-(2-carboxyethoxy)ethoxy]ethoxy}ethoxy)ethylcarbamoyl]propylcarbamoyl}nonadecanoic acid tert-butyl ester (0.68 g crude solution in THF 40 mL, 0.82 mmol) and TSTU (0.296 g, 0.982 mmol) were mixed, pH was adjusted by addition of DIPEA (0.171 mL) and the resulting mixture was stirred at RT overnight. This resulted in a clear solution which was evaporated to dryness and treated with diethylether, which afforded 1.2 g of the title compound as a waxy mass which was used without further purification.

MS (electrospray): m/z 816 (M−2 tBu) (calcd. 816).

Coupling of 19-(1-tert-butoxycarbonyl-3-{2-[2-(2-{2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonyl)ethoxy]-ethoxy}ethoxy)ethoxy]ethylcarbamoyl}propylcarbamoyl)nonadecanoic acid tert-butyl ester to A14E, A22K, B25H, B29R, desB30 Human Insulin:

A14E, A22K, B25H, B29R, desB30 human insulin (0.3 g, 0.052 mmol) was dissolved in a mixture of acetonitrile (4 mL) and Na₂CO₃ (0.1 M, 10 mL), pH was adjusted to 10.6 with NaOH (1 M). 19-(1-tert-Butoxycarbonyl-3-{2-[2-(2-{2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonyl)ethoxy]ethoxy}ethoxy)ethoxy]-ethylcarbamoyl}propylcarbamoyl)nonadecanoic acid tert-butyl ester (0.048 g, 0.052 mmol) in acetonitrile (4 mL) was added to the solution, and the mixture was gently stirred at RT for 1 h. 5 drops of methylamine solution (40% in MeOH) was added and the mixture was stirred for further 5 min. Then acetic acid (glacial, 5 mL) was added in one portion and the resulting mixture was purified by preparative HPLC (C18, 3 cm column, gradient 0-7 min 20% acetonitrile, 7-32 min 20-100% acetonitrile, 32-37 min 100% acetonitrile).

Pure fractions were pooled and freeze dried.

The resulting compound was dissolved in TFA (30 mL) and stirred at RT for 2 h, then acetonitrile (20 mL) and water (20 mL) were added and the mixture was purified by preparative HPLC (C18, 3 cm column, gradient 0-7 min 20% acetonitrile, 7-32 min 20-60% acetonitrile,32-37 min 60% acetonitrile).

Pure fractions were pooled and freeze dried.

Repurification by HPLC (C18, 3 cm column, gradient 0-7 min 25% acetonitrile, 7-47 min 25-60% acetonitrile, 47-52 min 60% acetonitrile), pure fractions were pooled and freeze dried.

This gave 13 mg final product.

MS (electrospray): m/z 6520 (calcd. 6520).

Example 10 General Procedure (A)

A18L, A22K(N^(ε)-Octadecandioyl-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]-acetyl), B29R, desB30 human insulin

MS (electrospray): m/z: 6578 (Calcd:6578).

Example 11 General Procedure (A)

A8H, A22K(N^(ε)-Octadecandioyl-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]-acetyl), B29R, desB30 human insulin

MALDI-TOF MS: m/z=6615 (Calcd.: 6615).

Example 12 General Procedure (A)

A22K(N^(ε)-Octadecandioyl-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl-γGlu), B29R, desB30 human insulin

MS (electrospray): m/z=1677 (m+4)/4.

Example 13 General Procedure (A)

A22K(N^(ε)-EicosanedioylγGlu-(3-{2-[2-(2-{2-[2-(2-amino-ethoxy)ethoxy]ethoxy}ethoxy)ethoxy]ethoxy}-propionyl)), B29R, desB30 human insulin

MS (electrospray): m/z=6653 (Calcd.: 6653).

Example 14 General Procedure (A)

A22K(N^(ε)-Octadecandioyl-γGlu-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl)), B29R, desB30 human insulin

MALDI-TOF MS: m/z=6535 (Calcd.: 6535)

Example 15 General Procedure (A)

A14E, A22K(N^(ε)-Octadecandioyl-γGlu-(3-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)ethoxy]ethoxy}ethoxy)ethoxy]propionyl)), B25H, B29R, desB30 human insulin

MS (electrospray): m/z=1667.9 (m+4)/4.

Example 16 General Procedure (A)

A14E, A22K(N^(ε)-Eicosanedioyl-γGlu-γGlu-(3-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)ethoxy]ethoxy}ethoxy)ethoxy]propionyl)), B25H, B29R, desB30 human insulin

MALDI-TOF MS: m/z=6824 (Calcd.: 6825)

Example 17 General Procedure (A)

A14E, A22K(N^(ε)-Octadecandioyl-γGlu-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl)-γGlu), B25H, B29R, desB30 human insulin

MALDI-TOF MS: m/z=6620 (Calcd.: 6621)

Example 18 General Procedure (A)

A22K(N^(ε)-Octadecandioyl-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B28E, B29R, desB30 human insulin

MALDI-TOF MS: m/z=6610 (Calcd.: 6610)

Example 19 General Procedure (A)

A22K(N^(ε)-Octadecandioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

MALDI-TOF MS: m/z=6724 (Calcd.: 6724)

The insulins in the following examples may be prepared using similar procedures:

Example 20

A14E, A22K(N^(ε)-[2-(2-[2-(2-[2-(Eicosanedioyl-γGlu)amino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl)), B25H, B29R, desB30 human insulin

Example 21

A22K(N^(ε)-Octadecandioyl-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl-γGlu), B29R, desB30 human insulin

Example 22

A22K(N^(ε)-Eicosanedioyl-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl-γGlu), B29R, desB30 human insulin

Example 23

A22K(N^(ε)-Octadecandioyl-(2-aminoethyl-PEG2000-ylacetyl)), B29R desB30 human insulin

Example 24

A22K(N^(ε)-Eicosanedioyl-(2-aminoethyl-PEG2000-ylacetyl)), B29R desB30 human insulin

Example 25

A22K(Nε-3-(3-{4-[3-(15-Carboxypentadecanoylamino)propoxy]butoxy}propylcarbamoyl)propionyl-γGlu), B29R desB30 human insulin

Example 26

A22K(Nε-3-(3-{4-[3-(17-Carboxyheptadecanoylamino)propoxy]butoxy}propylcarbamoyl)propionyl-γGlu), B29R desB30 human insulin

Example 27

A22K(N^(ε)-Tetradecandioyl-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl-γGlu), B29R, desB30 human insulin

Example 28

A8H, A22K(N^(ε)-Octadecandioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 29

A18L, A22K(N^(ε)-Octadecandioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)-ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 30

A22K(N^(ε)-Octadecandioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 31

A8H, A22K(N^(ε)-Octadecandioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 32

A18L, A22K(N^(ε)-Octadecandioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 33

A8H, A22K(N^(ε)-Eicosanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 34

A8H, A22K(N^(ε)-Eicosanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)-ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 35

A18L, A22K(N^(ε)-Eicosanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 36

A22K(N^(ε)-Eicosanedloyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 37

A8H, A22K(N^(ε)-Eicosanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 38

A18L, A22K(N^(ε)-Eicosanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 39

A8H, A22K(N^(ε)-Hexadecanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 40

A8H, A22K(N^(ε)-Hexadecanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 41

A18L, A22K(N^(ε)-Hexadecanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 42

A22K(N^(ε)-Hexadecanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 43

A8H, A22K(N^(ε)-Hexadecanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 44

A18L, A22K(N^(ε)-Hexadecanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 45

A22K(N^(ε)-Hexadecanedioyl-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 46

A8H, A22K(N^(ε)-Hexadecanedioyl-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 47

A18L, A22K(N^(ε)-Hexadecanedioyl-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin

Example 48 Insulin Receptor Binding of the Insulin Derivatives of this Invention

The affinity of the acylated insulin analogues of this invention for the human insulin receptor is determined by a SPA assay (Scintillation Proximity Assay) microtiterplate antibody capture assay. SPA-PVT antibody-binding beads, anti-mouse reagent (Amersham Biosciences, Cat No. PRNQ0017) are mixed with 25 ml of binding buffer (100 mM HEPES pH 7.8; 100 mM sodium chloride, 10 mM MgSO₄, 0.025% Tween-20). Reagent mix for a single Packard Optiplate (Packard No. 6005190) is composed of 2.4 pl of a 1:5000 diluted purified recombinant human insulin receptor (either with or without exon 11), an amount of a stock solution of A14Tyr[¹²⁵I]-human insulin corresponding to 5000 cpm per 100 μl of reagent mix, 12 μl of a 1:1000 dilution of F12 antibody, 3 ml of SPA-beads and binding buffer to a total of 12 ml. A total of 100 μl reagent mix is then added to each well in the Packard Optiplate and a dilution series of the insulin derivative is made in the Optiplate from appropriate samples. The samples are then incubated for 16 hours while gently shaken. The phases are the then separated by centrifugation for 1 min and the plates counted in a Topcounter. The binding data were fitted using the nonlinear regression algorithm in the GraphPad Prism 2.01 (GraphPad Software, San Diego, Calif.).

Insulin Receptor Affinities of Selected Insulins of the Invention

Insulin receptor affinities of selected insulins of the invention: IR-A IR-A Parent Insulin affinity affinity (all desB30 Ex. No. (0% HSA) (4.5% HSA) Modification human insulin)  100%  100% desB30 human insulin  102% ND A22K, B29R 24.7% ND A14E, A22K, B25H, B29R  9.2% 0.16% C18-gGlu desB30  168%  2.8% C18-gGlu A22K, B29R 4  161% 14.0% C18-gGlu- A22K, B29R OEG-OEG 19 ND   30% C18-gGlu- A22K, B29R OEG-OEG-OEG  149%  1.4% C20-gGlu A22K, B29R 6  148% 14.9% C20-gGlu- A22K, B29R OEG-OEG 18  162% 13.4% C18-gGlu- A22K, B28E, OEG-OEG B29R 10  339% 23.6% C18-gGlu- A18L, A22K, OEG-OEG B29R 11  417%   63% C18-gGlu- A8H, A22K, OEG-OEG B29R 7 22.4%  5.0% C18-gGlu- A14E, A22K, OEG-OEG B25H, B29R 8  132% ND C18-gGlu- A22K OEG-OEG 12  190% ND C18-gGlu- A22K, B29R OEG-OEG-gGlu 14  145%   27% C18-gGlue-PEG3 A22K, B29R 17   23% ND C18-gGlu- A14E, A22K, PEG3-gGlu B25H, B29R 15   28% ND C18-gGlu-PEG7 A14E, A22K, B25H, B29R 9 ND  5.2% C20-gGlu-PEG3 A14E, A22K, B25H, B29R 13  175% 10.9% C20-gGlu-PEG5 A14E, A22K, B25H, B29R 16   25% ND C20-gGlu- A14E, A22K, gGlu-PEG7 B25H, B29R

Example 49 Blood Glucose Lowering Effect After i.v. Bolus Injection in Rat of the Insulin Derivatives of this Invention

Male Wistar rats, 200-300 g, fasted for 18 h, is anesthetized using either Hypnorm-Dormicum s.c. (1.25 mg/ml Dormicum, 2.5 mg/ml fluanisone, 0.079 mg/ml fentanyl citrate) 2 ml/kg as a priming dose (to timepoint −30 min prior to test substance dosing) and additional 1 ml/kg every 20 minutes.

The animals are dosed with an intravenous injection (tail vein), 1 ml/kg, of control and test compounds (usual dose range 0.125-20 nmol/kg). Blood samples for the determination of whole blood glucose concentration are collected in heparinized 10 μl glass tubes by puncture of the capillary vessels in the tail tip to time −20 min and 0 min (before dosing), and to time 10, 20, 30, 40, 60, 80, 120, and 180 min after dosing. Blood glucose concentrations are measured after dilution in analysis buffer by the immobilized glucose oxidase method using an EBIO Plus autoanalyzer (Eppendorf, Germany). Mean plasma glucose concentrations courses (mean±SEM) are made for each dose and each compound.

Example 50 Potency of the Acylated Insulin Analogues of this Invention Relative to Human Insulin

Sprague Dawley male rats weighing 238-383 g on the experimental day are used for the clamp experiment. The rats have free access to feed under controlled ambient conditions and are fasted overnight (from 3 pm) prior to the clamp experiment.

Experimental Protocol:

The rats are acclimatized in the animal facilities for at least 1 week prior to the surgical procedure. Approximately 1 week prior to the clamp experiment, Tygon catheters are inserted under halothane anaesthesia into the jugular vein (for infusion) and the carotid artery (for blood sampling) and exteriorised and fixed on the back of the neck. The rats are given Streptocilin vet. (Boehringer Ingelheim; 0.15 ml/rat, i.m.) post-surgically and placed in an animal care unit (25° C.) during the recovery period. In order to obtain analgesia, Anorphin (0.06 mg/rat, s.c.) is administered during anaesthesia and Rimadyl (1.5 mg/kg, s.c.) is administered after full recovery from the anaesthesia (2-3 h) and again once daily for 2 days.

At 7 am on the experimental day overnight fasted (from 3 pm the previous day) rats are weighed and connected to the sampling syringes and infusion system (Harvard 22 Basic pumps, Harvard, and Perfectum Hypodermic glass syringe, Aldrich) and then placed into individual clamp cages where they rest for ca. 45 min before start of experiment. The rats are able to move freely on their usual bedding during the entire experiment and have free access to drinking water. After a 30 min basal period during which plasma glucose levels were measured at 10 min intervals, the insulin derivative to be tested and human insulin (one dose level per rat, n=6-7 per dose level) are infused (i.v.) at a constant rate for 300 min. Plasma glucose levels are measured at 10 min intervals throughout and infusion of 20% aqueous glucose is adjusted accordingly in order to maintain euglyceamia. Samples of re-suspended erythrocytes are pooled from each rat and returned in about ½ ml volumes via the carotid catheter.

On each experimental day, samples of the solutions of the individual insulin derivatives to be tested and the human insulin solution are taken before and at the end of the clamp experiments and the concentrations of the peptides are confirmed by HPLC. Plasma concentrations of rat insulin and C-peptide as well as of the insulin derivative to be tested and human insulin are measured at relevant time points before and at the end of the studies. Rats are killed at the end of experiment using a pentobarbital overdose.

Example 51 Pulmonary Delivery of Insulin Derivatives to Rats

The test substance will be dosed pulmonary by the drop instillation method. In brief, male Wistar rats (app.250 g) are anaesthesized in app. 60 ml fentanyl/dehydrodenzperidol/-dormicum given as a 6.6 ml/kg sc priming dose and followed by 3 maintenance doses of 3.3 ml/kg sc with an interval of 30 min. Ten minutes after the induction of anaesthesia, basal samples are obtained from the tail vein (t=−20 min) followed by a basal sample immediately prior to the dosing of test substance (t=0). At t=0, the test substance is dosed intra tracheally into one lung. A special cannula with rounded ending is mounted on a syringe containing the 200 ul air and test substance (1 ml/kg). Via the orifice, the cannula is introduced into the trachea and is forwarded into one of the main bronchi—just passing the bifurcature. During the insertion, the neck is palpated from the exterior to assure intratracheal positioning. The content of the syringe is injected followed by 2 sec pause. Thereafter, the cannula is slowly drawn back. The rats are kept anaesthesized during the test (blood samples for up to 4 or 8 hrs) and are euthanized after the experiment.

Sequence Listings

SEQ ID NO:1 is the A1-A21 chain of examples 1-6, 8, 12-14, 18 and 19. SEQ ID NO:2 is the B1-B29 chain of examples 1-6, 10-14 and 19. SEQ ID NO:3 is the A1-A21 chain of examples 7, 9 and 15-17. SEQ ID NO:4 is the B1-B29 chain of examples 7, 9 and 15-17. SEQ ID NO:5 is the A1-A21 chain of example 10. SEQ ID NO:6 is the A1-A21 chain of example 11. SEQ ID NO:7 is the B1-B29 chain of examples 18. SEQ ID NO:8 is the B1-B28 chain of example 8. 

1. An acylated insulin analogue wherein the insulin analogue comprises a lysine residue connected C-terminally to the A21 amino acid residue or a peptide residue of up to 4 amino acid residues comprising a lysine residue which peptide residue is connected C-terminally to the A21 amino acid residue, characterized in that an acyl moiety comprising an alkylene glycol moiety is attached to the lysine residue in the A22 position or attached to a lysine residue present in the peptide residue that is attached to the C terminal end of the A21 amino acid residue and wherein there is only one lysine (K, Lys) in the insulin analogue.
 2. An acylated insulin analogue, according to claim 1, wherein the acyl moiety has the general formula I: Acy-AA1_(n)-AA2_(m)-AA3_(p)-  (I), wherein n is 0 or an integer in the range 1-3, m is 0 or an integer in the range 1-6, p is 1, 2 or 3, Acy is a fatty acid or a fatty diacid comprising about 8-24 carbon atoms from which, formally, a hydroxy group has been removed from the carboxy group of the fatty acid or from one of the carboxy groups of the fatty diacid, AA1 is a neutral cyclic amino acid from which, formally, a hydrogen atom has been removed from the amino group and a hydroxy group has been removed from the carboxy group, AA2 is an acidic amino acid from which, formally, a hydrogen atom has been removed from the amino group and a hydroxy group has been removed from the carboxy group, AA3 is a neutral, alkyleneglycol-containing amino acid from which, formally, a hydrogen atom has been removed from the amino group and a hydroxy group has been removed from the carboxy group, the order by which AA1, AA2 and AA3 appears in the formula can be interchanged independently, the connections between Acy, AA1, AA2 and/or AA3 are amide (peptide) bonds, and attachment to the parent insulin can be from the C-terminal end of a AA1, AA2, or AA3 residue in the acyl moiety of the formula (I) or from one of the side-chain(s) of an AA2 residue present in the moiety of formula (I).
 3. An acylated insulin analogue, according to any one of the preceding, possible claims, wherein AA1 is selected from:

from which, formally, a hydrogen atom has been removed from the amino group and a hydroxy group has been removed from the carboxy group, and wherein q is 0, 1, 2, 3 or
 4. 4. An acylated insulin analogue, according to the preceding claim, wherein AA1 is

from which, formally, a hydrogen atom has been removed from the amino group and a hydroxy group has been removed from the carboxy group.
 5. An acylated insulin analogue, according to any one of the preceding, possible claims, wherein AA2 is selected from γGlu, βAsp or any of the following compounds:

from which, formally, a hydrogen atom has been removed from the amino group and a hydroxy group has been removed from the carboxy group, and wherein the arrows indicate the attachment point to the amino group of AA1, AA2 or AA3.
 6. An acylated insulin analogue, according to any one of the preceding, possible claims which is any of the following comoiunds: A22K(N^(ε)-hexadecandioyl-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl-γGlu), B29R, desB30 human insulin; A22K(N^(ε)-hexadecandioyl-(2-aminoethyl-PEG2000-yl-acetyl)), B29R desB30 human insulin; A22K(N^(ε)-3-(3-{4-[3-(5-carboxypentanoylamino)propoxy]butoxy}propylcarbamoyl)propionyl-γGlu), B29R, desB30 human insulin; A22K(N^(ε)-[2(2-[2-(2-[2-(octadecandioyl-γGlu)amino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]), B29R, desB30 human insulin; A22K(N^(ε)-3-(3-{4-[3-(13-carboxytridecanoylamino)propoxy]butoxy}propylcarbamoyl)propionyl-γGlu), B29R, desB30 human insulin; A22K(N^(ε)-[2-(2-[2-(2-[2-(eicosanedioyl-γGlu)amino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]), B29R, desB30 human insulin; A14E, A22K(N^(ε)-[2-(2-[2-(2-[2-(octadecandioyl-γGlu)amino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]), B25H, B29R, desB30 human insulin; A22K(N^(ε)-octadecandioyl-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), desB29, desB30 human insulin; A14E, A22K(N^(ε)-eicosanedioyl-γGlu-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl)), B25H, B29R, desB30 human insulin; A18L, A22K(N^(ε)-octadecandioyl-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A8H, A22K(N^(ε)-octadecandioyl-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A22K(N^(ε)-octadecandioyl-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl-γGlu), B29R, desB30 human insulin; A22K(N^(ε)-eicosanedioyl-γGlu-(3-{2-[2-(2-{2-[2-(2-amino-ethoxy)ethoxy]ethoxy}ethoxy)ethoxy]ethoxy}propionyl)), B29R, desB30 human insulin; A22K(N^(ε)-octadecandioyl-γGlu-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl)), B29R, desB30 human insulin; A14E, A22K(N^(ε)-octadecandioyl-γGlu-(3-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)ethoxy]ethoxy}ethoxy)ethoxy]propionyl)), B25H, B29R, desB30 human insulin; A14E, A22K(N^(ε)-eicosanedioyl-γGlu-γGlu-(3-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)ethoxy]ethoxy}ethoxy)ethoxy]propionyl)), B25H, B29R, desB30 human insulin; A14E, A22K(N^(ε)-octadecandioyl-γGlu-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl)-γGlu), B25H, B29R, desB30 human insulin; A22K(N^(ε)-octadecandioyl-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B28E, B29R, desB30 human insulin; 22K(N^(ε)-octadecandioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A14E, A22K(N^(ε)-[2-(2-[2-(2-[2-(eicosanedioyl-γGlu)amino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]), B25H, B29R, desB30 human insulin; A22K(N^(ε)-octadecandioyl-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl-γGlu), B29R, desB30 human insulin; A22K(N^(ε)-eicosanedioyl-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl-γGlu), B29R, desB30 human insulin; A22K(N^(ε)-octadecandioyl-(2-aminoethyl-PEG2000-ylacetyl)), B29R desB30 human insulin; A22K(N^(ε)-eicosanedioyl-(2-aminoethyl-PEG2000-yl-acetyl)), B29R desB30 human insulin; A22K(Nε-3-(3-{4-[3-(15-carboxypentadecanoylamino)propoxy]butoxy}propylcarbamoyl)propionyl-γGlu), B29R desB30 human insulin; A22K(Nε-3-(3-{4-[3-(17-carboxyheptadecanoylamino)propoxy]butoxy}propylcarbamoyl)propionyl-γGlu), B29R desB30 human insulin; A22K(N^(ε)-tetradecandioyl-(3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionyl-γGlu), B29R, desB30 human insulin; A8H, A22K(N^(ε)-octadecandioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), 829R, desB30 human insulin; A18L, A22K(N^(ε)-octadecandioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A22K(N^(ε)-octadecandioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A8H, A22K(N^(ε)-octadecandioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A18L, A22K(N^(ε)-octadecandioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A8H, A22K(N^(ε)-eicosanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A8H, A22K(N^(ε)-eicosanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A18L, A22K(N^(ε)-eicosanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A22K(N^(ε)-eicosanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A8H, A22K(N^(ε)-eicosanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A18L, A22K(N^(ε)-eicosanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A8H, A22K(N^(ε)-hexadecanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A8H, A22K(N^(ε)-hexadecanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetyl-amino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A18L, A22K(N^(ε)-hexadecanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A22K(N^(ε)-hexadecanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A8H, A22K(N^(ε)-hexadecanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]-acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A18L, A22K(N^(ε)-hexadecanedioyl-γGlu-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A22K(N^(ε)-hexadecanedioyl-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin; A8H, A22K(N^(ε)-hexadecanedioyl-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin and A18L, A22K(N^(ε)-hexadecanedioyl-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), B29R, desB30 human insulin. 